Macropinocytosing human anti-CD46 antibodies and targeted cancer therapeutics

ABSTRACT

In various embodiments human anti-CD46 antibodies that are internalizing and enter tumor cells via the macropinocytosis pathway are provided, as well as antibody-drug conjugates (ADCs) developed from these antibodies for diagnostic and/or therapeutic targeting of CD46-overexpressing tumors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. 371 National Phase of PCT/US2015/049492,filed on Sep. 10, 2015, which claims benefit of and priority to U.S.Ser. No. 62/049,973, filed on Sep. 12, 2014, all of which areincorporated herein by reference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under grant nos. R01CA118919, R01 CA129491 and R01 CA171315 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file,“UCSF-P037US_ST25.txt” created on Aug. 23, 2017 and having a size of98,700 bytes. The contents of the text file are incorporated byreference herein in their entirety.

BACKGROUND

Due to ease of accessibility, tumor cell surface antigens are valuabletargets for therapeutic development. The epitope space at the cellsurface is highly complex. Relevant antigens may include glycosylatedproteins and other post-translationally modified products that may notbe readily predicted from studies of genomic copy number or mRNAexpression levels (Liu et al. (2004) Cancer Res. 64: 704-710; Kobata andAmano (2005) Immunol. Cell Biol. 83: 429-439; Birkle et al. (2003)Biochimie (Paris) 85: 455-463; Hakomori (2001) Adv. Exp. Med. Biol. 491:369-402; Hanisch, F. G. (2001) O-Glycosylation of the mucin type. Biol.Chem. 382, 143-1 49; Ugorski and Laskowska (2002) Acta Biochim. Pol. 49:303-311).

Identification of tumor cell surface epitopes allows the production ofantibodies to achieve specific binding to neoplastic cells, an abilitythat can be utilized in applications such as induction ofantibody-dependent cell cytotoxicity (see, e.g., Clynes et al. (2000)Nat. Med. 6: 443-446), or inhibition of signaling pathways involved intumor cell migration, growth, and survival (see, e.g., McWhirter et al.(2006) Proc. Natl. Acad. Sci., USA, 103: 1041-1 046; Fuh et al. (2006)J. Biol. Chem. 281: 6625-6631). In addition, antibodies targetinginternalizing tumor epitopes can be exploited to achieve efficient andspecific intracellular delivery of cytotoxins, cytostatic agents,chemotherapeutic drugs and/or other tumor-modulating agents (see, e.g.,Liu et al. (2004) Cancer Res. 64: 704-710; Nielsen et al. (2002)Biochim. Biophys. Acta 1591: 109-118; Pirollo et al. (2006) Hum. GeneTher. 17: 117-124; Song et al. (2005) Nat. Biotechnol. 23:709-717; Liuet al. (2002) J. Mol. Biol. 315: 1063-1073).

Phage antibody display has been widely used to develop cancer-specificantibodies (see, e.g., Liu et al. (2004) Cancer Res. 64: 704-710; Liuand Marks (2000) Anal. Biochem. 286: 119-128; 15. Marks et al. (1992)Biotechnology (N.Y.) 10: 779-783; Marks et al. (1991) J. Mol. Biol. 222:581-597; Marks et al. (1992) J. Biol. Chem. 267: 16007-16010; Sharon etal. (2005) J. Cell. Biochem. 96: 305-313; Silacci et al. (2005)Proteomics 5: 2340-2350; Gao et al. (2003) J. Immunol. Methods 274:185-197; Lekkerkerker and Logtenberg (1999) J. Immunol. Meth., 231:53-63; de Kruif et al. (1995) Proc. Natl. Acad. Sci., USA, 92:3938-3942; Pini et al. (1998) J. Biol. Chem. 273: 21 769-21 776). Acombinatorial phage antibody library serves as a source of random shaperepertoire that can be used to probe neoplastic variations on thesurface of cancer cells (see, e.g., Liu et al. (2004) Cancer Res. 64:704-710; Geuijen et al. (2005) Eur. J. Cancer 41: 178-187; Poul et al.(2000) J. Mol. Biol. 301: 1149-1161; Cai and Garen (1995) Proc. Natl.Acad. Sci., USA, 92: 6537-6541). Selecting phage antibody librariesdirectly on cancer cell lines enables the identification oftumor-targeting antibodies without prior knowledge of target antigenssee, e.g., (Liu et al. (2004) Cancer Res. 64: 704-710; Gao et al. (2003)J. Immunol. Methods 274: 185-197; Geuijen et al. (2005) Eur. J. Cancer41: 178-187; Poul et al. (2000) J. Mol. Biol. 301: 1149-1161).

Although numerous antibodies have been found by this approach, thescreening process against cell lines does not provide an ideal pictureas to how specific these antibodies will be to actual cancer cells inpatient populations. Nor does it necessarily provide an indication ofwhether or not the antibodies will internalize in vivo.

SUMMARY

In various embodiments human anti-CD46 antibodies that are internalizingand enter tumor cells via the macropinocytosis pathway are provided, aswell as antibody-drug conjugates (ADCs) developed from these antibodiesfor diagnostic and/or therapeutic targeting of CD46-overexpressingtumors.

Various embodiments contemplated herein may include, but need not belimited to, one or more of the following:

Embodiment 1: An isolated human antibody that specifically binds CD46and is internalized into a cell expressing or overexpressing CD46,wherein: said antibody is an antibody that specifically binds cells thatexpress or overexpress a CD46, wherein said antibody specifically bindsan epitope bound by one or more antibodies selected from the groupconsisting of YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY, YS6, YS1, YS3,YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/orUA8kappa; and said antibody is internalized into said cell viamacropinocytosis.

Embodiment 2: The antibody of embodiment 1, wherein said antibody bindsdomain 1 and/or domain 2 of CD46.

Embodiment 3: The antibody of embodiments 1 or 2, wherein said antibodydoes not bind domain 3 and/or domain 4 of CD46.

Embodiment 4: The antibody according to any one of embodiments 1-3,wherein said cells that express or overexpress a CD46 are cancer cells.

Embodiment 5: The antibody according to any one of embodiments 1-4,wherein said cells that express or overexpress a CD46 are prostatecancer cells.

Embodiment 6: The antibody of embodiment 5, wherein said antibody bindscells of a cell line selected from the group consisting of DU145 cells,PC3 cells, and LnCaP cells.

Embodiment 7: The antibody according to any one of embodiments 1-6,wherein said antibody binds to a prostate tumor cell with an affinity(K_(D)) of at least about 5-10 nM when measured on live prostate tumorcells by FACS.

Embodiment 8: The antibody of embodiment 7, wherein said antibody bindsto a prostate tumor cell with an affinity (K_(D)) of at least about 3 nMwhen measured on live prostate tumor cells by FACS.

Embodiment 9: The antibody according to any one of embodiments 1-8,wherein said antibody is a substantially intact immunoglobulin.

Embodiment 10: The antibody of embodiment 9, wherein said antibodycomprises an IgA, IgE, or IgG.

Embodiment 11: The antibody of embodiment 9, wherein said antibodycomprises an IgG1.

Embodiment 12: The antibody according to any one of embodiments 1-8,wherein said antibody is an antibody fragment that specifically bindscells that express or overexpress a CD46.

Embodiment 13: The antibody of embodiment 12, wherein said antibody isan antibody fragment selected from the group consisting of Fv, Fab,(Fab′)₂, (Fab′)₃, IgGΔCH2, and a minibody.

Embodiment 14: The antibody according to any one of embodiments 1-8,wherein said antibody is a single chain antibody.

Embodiment 15: The antibody of embodiment 14, wherein the VL region ofsaid antibody is attached to the VH region of said antibody by an aminoacid linker ranging in length from about 3 amino acids up to about 15amino acids.

Embodiment 16: The antibody of embodiment 14, wherein the VL region ofsaid antibody is attached to the VH region of said antibody by an aminoacid linker selected from the group consisting of GGGGS GGGGS GGGGS (SEQID NO:67), GGGGS GGGGS (SEQ ID NO:68), GGGGS (SEQ ID NO:69), GS GGGGSGGGGS GGS GGGGS (SEQ ID NO:70), SGGGGS (SEQ ID NO:71), GGGS (SEQ IDNO:72), VPGV (SEQ ID NO:73), VPGVG (SEQ ID NO:74), GVPGVG (SEQ IDNO:75), GVG VP GVG (SEQ ID NO:76), VP GVG VP GVG (SEQ ID NO:77), GGSSRSS(SEQ ID NO:78), and GGSSRSSSSGGGGSGGGG (SEQ ID NO:79).

Embodiment 17: The antibody according to any one of embodiments 1-16,wherein said antibody competes with YS5 for binding at CD46.

Embodiment 18: The antibody according to any one of embodiments 1-16,wherein said antibody competes with YS5F for binding at CD46.

Embodiment 19: The antibody according to any one of embodiments 1-16,wherein said antibody competes with YS5v1D for binding at CD46.

Embodiment 20: The antibody according to any one of embodiments 1-16,wherein said antibody competes with SB1HGNY for binding at CD46.

Embodiment 21: The antibody according to any one of embodiments 1-16,wherein said antibody competes with YS12 for binding at CD46.

Embodiment 22: The antibody according to any one of embodiments 1-16,wherein said antibody competes with 3G7RY for binding at CD46.

Embodiment 23: The antibody according to any one of embodiments 1-16,wherein said antibody competes with YS6 for binding at CD46.

Embodiment 24: The antibody according to any one of embodiments 1-16,wherein said antibody competes with YS1 for binding at CD46.

Embodiment 25: The antibody according to any one of embodiments 1-16,wherein said antibody competes with YS3 for binding at CD46.

Embodiment 26: The antibody according to any one of embodiments 1-16,wherein said antibody competes with YS4 for binding at CD46.

Embodiment 27: The antibody according to any one of embodiments 1-16,wherein said antibody competes with YS8 for binding at CD46.

Embodiment 28: The antibody according to any one of embodiments 1-16,wherein said antibody competes with YS7 for binding at CD46.

Embodiment 29: The antibody according to any one of embodiments 1-16,wherein said antibody competes with YS9 for binding at CD46.

Embodiment 30: The antibody according to any one of embodiments 1-16,wherein said antibody competes with YS10 for binding at CD46.

Embodiment 31: The antibody according to any one of embodiments 1-16,wherein said antibody competes with YS11 for binding at CD46.

Embodiment 32: The antibody according to any one of embodiments 1-16,wherein said antibody competes with 3G7HY for binding at CD46.

Embodiment 33: The antibody according to any one of embodiments 1-16,wherein said antibody competes with 3G7NY for binding at CD46.

Embodiment 34: The antibody according to any one of embodiments 1-16,wherein said antibody competes with 3G7 for binding at CD46.

Embodiment 35: The antibody according to any one of embodiments 1-16,wherein said antibody competes with SB2 for binding at CD46.

Embodiment 36: The antibody according to any one of embodiments 1-16,wherein said antibody competes with 2C8 for binding at CD46.

Embodiment 37: The antibody according to any one of embodiments 1-16,wherein said antibody competes with UA8kappa for binding at CD46.

Embodiment 38: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3,and/or VL CDR1, and/or VL CDR2, and/or VL CDR3 of an antibody selectedfrom the group consisting of YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY,YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2,2C8, and UA8kappa.

Embodiment 39: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the YS5 antibody.

Embodiment 40: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the YS5 antibody.

Embodiment 41: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the YS5 antibody.

Embodiment 42: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the YS5antibody.

Embodiment 43: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of the YS5antibody.

Embodiment 44: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the YS5antibody and the variable heavy (VH) chain of the YS5 antibody.

Embodiment 45: The antibody of embodiment 1, wherein said antibody is ahuman YS5 scFv.

Embodiment 46: The antibody of embodiment 1, wherein said antibody is ahuman YS5 IgG.

Embodiment 47: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the YS5F antibody.

Embodiment 48: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the YS5F antibody.

Embodiment 49: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the YS5F antibody.

Embodiment 50: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of theYS5F antibody.

Embodiment 51: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of theYS5F antibody.

Embodiment 52: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of theYS5F antibody and the variable heavy (VH) chain of the YS5F antibody.

Embodiment 53: The antibody of embodiment 1, wherein said antibody is ahuman YS5F scFv.

Embodiment 54: The antibody of embodiment 1, wherein said antibody is ahuman YS5F IgG.

Embodiment 55: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the YS5F antibody.

Embodiment 56: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the YS5F antibody.

Embodiment 57: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the YS5F antibody.

Embodiment 58: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of theYS5F antibody.

Embodiment 59: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of theYS5F antibody.

Embodiment 60: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of theYS5F antibody and the variable heavy (VH) chain of the YS5F antibody.

Embodiment 61: The antibody of embodiment 1, wherein said antibody is ahuman YS5F scFv.

Embodiment 62: The antibody of embodiment 1, wherein said antibody is ahuman YS5F IgG.

Embodiment 63: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the YS5V1D antibody.

Embodiment 64: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the YS5V1D antibody.

Embodiment 65: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the YS5V1D antibody.

Embodiment 66: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of theYS5V1D antibody.

Embodiment 67: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of theYS5V1D antibody.

Embodiment 68: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of theYS5V1D antibody and the variable heavy (VH) chain of the YS5V1Dantibody.

Embodiment 69: The antibody of embodiment 1, wherein said antibody is ahuman YS5V1D scFv.

Embodiment 70: The antibody of embodiment 1, wherein said antibody is ahuman YS5V1D IgG.

Embodiment 71: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the SB1HGNY antibody.

Embodiment 72: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the SB1HGNY antibody.

Embodiment 73: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the SB1HGNY antibody.

Embodiment 74: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of theSB1HGNY antibody.

Embodiment 75: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of theSB1HGNY antibody.

Embodiment 76: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of theSB1HGNY antibody and the variable heavy (VH) chain of the SB1HGNYantibody.

Embodiment 77: The antibody of embodiment 1, wherein said antibody is ahuman SB1HGNY scFv.

Embodiment 78: The antibody of embodiment 1, wherein said antibody is ahuman SB1HGNY IgG.

Embodiment 79: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the YS12 antibody.

Embodiment 80: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the YS12 antibody.

Embodiment 81: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the YS12 antibody.

Embodiment 82: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of theYS12 antibody.

Embodiment 83: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of theYS12 antibody.

Embodiment 84: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of theYS12 antibody and the variable heavy (VH) chain of the YS12 antibody.

Embodiment 85: The antibody of embodiment 1, wherein said antibody is ahuman YS12 scFv.

Embodiment 86: The antibody of embodiment 1, wherein said antibody is ahuman YS12 IgG.

Embodiment 87: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the 3G7RY antibody.

Embodiment 88: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the 3G7RY antibody.

Embodiment 89: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the 3G7RY antibody.

Embodiment 90: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the3G7RY antibody.

Embodiment 91: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of the3G7RY antibody.

Embodiment 92: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the3G7RY antibody and the variable heavy (VH) chain of the 3G7RY antibody.

Embodiment 93: The antibody of embodiment 1, wherein said antibody is ahuman 3G7RY scFv.

Embodiment 94: The antibody of embodiment 1, wherein said antibody is ahuman 3G7RY IgG.

Embodiment 95: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the YS6 antibody.

Embodiment 96: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the YS6 antibody.

Embodiment 97: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the YS6 antibody.

Embodiment 98: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the YS6antibody.

Embodiment 99: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of the YS6antibody.

Embodiment 100: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the YS6antibody and the variable heavy (VH) chain of the YS6 antibody.

Embodiment 101: The antibody of embodiment 1, wherein said antibody is ahuman YS6 scFv.

Embodiment 102: The antibody of embodiment 1, wherein said antibody is ahuman YS6 IgG.

Embodiment 103: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the YS1 antibody.

Embodiment 104: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the YS1 antibody.

Embodiment 105: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the YS1 antibody.

Embodiment 106: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the YS1antibody.

Embodiment 107: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of the YS1antibody.

Embodiment 108: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the YS1antibody and the variable heavy (VH) chain of the YS1 antibody.

Embodiment 109: The antibody of embodiment 1, wherein said antibody is ahuman YS1 scFv.

Embodiment 110: The antibody of embodiment 1, wherein said antibody is ahuman YS1 IgG.

Embodiment 111: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the YS3 antibody.

Embodiment 112: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the YS3 antibody.

Embodiment 113: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the YS3 antibody.

Embodiment 114: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the YS3antibody.

Embodiment 115: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of the YS3antibody.

Embodiment 116: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the YS3antibody and the variable heavy (VH) chain of the YS3 antibody.

Embodiment 117: The antibody of embodiment 1, wherein said antibody is ahuman YS3 scFv.

Embodiment 118: The antibody of embodiment 1, wherein said antibody is ahuman YS3 IgG.

Embodiment 119: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the YS4 antibody.

Embodiment 120: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the YS4 antibody.

Embodiment 121: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the YS4 antibody.

Embodiment 122: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the YS4antibody.

Embodiment 123: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of the YS4antibody.

Embodiment 124: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the YS4antibody and the variable heavy (VH) chain of the YS4 antibody.

Embodiment 125: The antibody of embodiment 1, wherein said antibody is ahuman YS4 scFv.

Embodiment 126: The antibody of embodiment 1, wherein said antibody is ahuman YS4 IgG.

Embodiment 127: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the YS8 antibody.

Embodiment 128: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the YS8 antibody.

Embodiment 129: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the YS8 antibody.

Embodiment 130: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the YS8antibody.

Embodiment 131: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of the YS8antibody.

Embodiment 132: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the YS8antibody and the variable heavy (VH) chain of the YS8 antibody.

Embodiment 133: The antibody of embodiment 1, wherein said antibody is ahuman YS8 scFv.

Embodiment 134: The antibody of embodiment 1, wherein said antibody is ahuman YS8 IgG.

Embodiment 135: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the YS7 antibody.

Embodiment 136: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the YS7 antibody.

Embodiment 137: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the YS7 antibody.

Embodiment 138: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the YS7antibody.

Embodiment 139: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of the YS7antibody.

Embodiment 140: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the YS7antibody and the variable heavy (VH) chain of the YS7 antibody.

Embodiment 141: The antibody of embodiment 1, wherein said antibody is ahuman YS7 scFv.

Embodiment 142: The antibody of embodiment 1, wherein said antibody is ahuman YS7 IgG.

Embodiment 143: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the YS9 antibody.

Embodiment 144: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the YS9 antibody.

Embodiment 145: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the YS9 antibody.

Embodiment 146: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the YS9antibody.

Embodiment 147: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of the YS9antibody.

Embodiment 148: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the YS9antibody and the variable heavy (VH) chain of the YS9 antibody.

Embodiment 149: The antibody of embodiment 1, wherein said antibody is ahuman YS9 scFv.

Embodiment 150: The antibody of embodiment 1, wherein said antibody is ahuman YS9 IgG.

Embodiment 151: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the YS10 antibody.

Embodiment 152: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the YS10 antibody.

Embodiment 153: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the YS10 antibody.

Embodiment 154: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of theYS10 antibody.

Embodiment 155: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of theYS10 antibody.

Embodiment 156: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of theYS10 antibody and the variable heavy (VH) chain of the YS10 antibody.

Embodiment 157: The antibody of embodiment 1, wherein said antibody is ahuman YS10 scFv.

Embodiment 158: The antibody of embodiment 1, wherein said antibody is ahuman YS10 IgG.

Embodiment 159: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the YS11 antibody.

Embodiment 160: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the YS11 antibody.

Embodiment 161: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the YS11 antibody.

Embodiment 162: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of theYS11 antibody.

Embodiment 163: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of theYS11 antibody.

Embodiment 164: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of theYS11 antibody and the variable heavy (VH) chain of the YS11 antibody.

Embodiment 165: The antibody of embodiment 1, wherein said antibody is ahuman YS scFv.

Embodiment 166: The antibody of embodiment 1, wherein said antibody is ahuman YS IgG.

Embodiment 167: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the 3G7HY antibody.

Embodiment 168: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the 3G7HY antibody.

Embodiment 169: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the 3G7HY antibody.

Embodiment 170: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the3G7HY antibody.

Embodiment 171: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of the3G7HY antibody.

Embodiment 172: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the3G7HY antibody and the variable heavy (VH) chain of the 3G7HY antibody.

Embodiment 173: The antibody of embodiment 1, wherein said antibody is ahuman 3G7HY scFv.

Embodiment 174: The antibody of embodiment 1, wherein said antibody is ahuman 3G7HY IgG.

Embodiment 175: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the 3G7NY antibody.

Embodiment 176: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the 3G7NY antibody.

Embodiment 177: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the 3G7NY antibody.

Embodiment 178: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the3G7NY antibody.

Embodiment 179: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of the3G7NY antibody.

Embodiment 180: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the3G7NY antibody and the variable heavy (VH) chain of the 3G7NY antibody.

Embodiment 181: The antibody of embodiment 1, wherein said antibody is ahuman 3G7NY scFv.

Embodiment 182: The antibody of embodiment 1, wherein said antibody is ahuman 3G7NY IgG.

Embodiment 183: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the 3G7 antibody.

Embodiment 184: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the 3G7 antibody.

Embodiment 185: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the 3G7 antibody.

Embodiment 186: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the 3G7antibody.

Embodiment 187: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of the 3G7antibody.

Embodiment 188: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the 3G7antibody and the variable heavy (VH) chain of the 3G7 antibody.

Embodiment 189: The antibody of embodiment 1, wherein said antibody is ahuman 3G7 scFv.

Embodiment 190: The antibody of embodiment 1, wherein said antibody is ahuman 3G7 IgG.

Embodiment 191: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the SB2 antibody.

Embodiment 192: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the SB2 antibody.

Embodiment 193: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the SB2 antibody.

Embodiment 194: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the SB2antibody.

Embodiment 195: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of the SB2antibody.

Embodiment 196: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the SB2antibody and the variable heavy (VH) chain of the SB2 antibody.

Embodiment 197: The antibody of embodiment 1, wherein said antibody is ahuman SB2 scFv.

Embodiment 198: The antibody of embodiment 1, wherein said antibody is ahuman SB2 IgG.

Embodiment 199: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the 2C8 antibody.

Embodiment 200: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the 2C8 antibody.

Embodiment 201: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the 2C8 antibody.

Embodiment 202: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the 2C8antibody.

Embodiment 203: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of the 2C8antibody.

Embodiment 204: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of the 2C8antibody and the variable heavy (VH) chain of the 2C8 antibody.

Embodiment 205: The antibody of embodiment 1, wherein said antibody is ahuman 2C8 scFv.

Embodiment 206: The antibody of embodiment 1, wherein said antibody is ahuman 2C8 IgG.

Embodiment 207: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, and/or VH CDR2, and/or VH CDR3of the UA8kappa antibody.

Embodiment 208: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VL CDR1, and/or VL CDR2, and/or VL CDR3of the UA8kappa antibody.

Embodiment 209: The antibody according to any one of embodiments 1-16,wherein said antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VLCDR2, and VL CDR3 of the UA8kappa antibody.

Embodiment 210: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of theUA8kappa antibody.

Embodiment 211: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable heavy (VH) chain of theUA8kappa antibody.

Embodiment 212: The antibody according to any one of embodiments 1-16,wherein said antibody comprises the variable light (VL) chain of theUA8kappa antibody and the variable heavy (VH) chain of the UA8kappaantibody.

Embodiment 213: The antibody of embodiment 1, wherein said antibody is ahuman UA8kappa scFv.

Embodiment 214: The antibody of embodiment 1, wherein said antibody is ahuman UA8kappa IgG.

Embodiment 215: An immunoconjugate including an antibody according toany one of embodiments 1-214 attached to an effector wherein saideffector is selected from the group consisting of a second antibody, adetectable label, a cytotoxin or cytostatic agent, a liposome containinga drug, a radionuclide, a drug, a prodrug, a viral particle, a cytokine,and a chelate.

Embodiment 216: The immunoconjugate of embodiment 215, wherein saidantibody is attached to a cytotoxin.

Embodiment 217: The immunoconjugate of embodiment 216, wherein saidantibody is attached to a cytotoxin selected from the group consistingof a Diphtheria toxin, a Pseudomonas exotoxin, a ricin, an abrin,saporin, and a thymidine kinase.

Embodiment 218: The immunoconjugate of embodiment 215, wherein saidantibody is attached to a cytotoxic and/or cytostatic drug.

Embodiment 219: The immunoconjugate of embodiment 216, wherein saidantibody is attached directly or through a linker to one or more of thefollowing: said drug a lipid or liposome containing said drug; apolymeric drug carrier including said drug; and a nanoparticle drugcarrier including said drug.

Embodiment 220: The immunoconjugate according to any one of embodiments218-219, wherein said drug is an anti-cancer drug.

Embodiment 221: The immunoconjugate according to any one of embodiments218-219, wherein said drug is selected from the group consisting of amicrotubule inhibitor, a DNA-damaging agents, and a polymeraseinhibitor.

Embodiment 222: The immunoconjugate of embodiment 221, wherein the drugcomprises a tubulin inhibitor.

Embodiment 223: The immunoconjugate of embodiment 222, wherein the drugcomprises a drug selected from the group consisting of an auristatin,Dolastatin-10, synthetic derivatives of the natural productDolastatin-10, and maytansine or a maytansine derivative.

Embodiment 224: The immunoconjugate of embodiment 222, wherein the drugcomprises a drug selected from the group consisting MonomethylauristatinF (MMAF), Auristatin E (AE), Monomethylauristatin E (MMAE), vcMMAE, andvcMMAF.

Embodiment 225: The immunoconjugate of embodiment 222, wherein the drugcomprises a maytansine selected from the group consisting of Mertansine(DM1), DM3, and DM4.

Embodiment 226: The immunoconjugate of embodiment 221, wherein the drugcomprises a DNA-damaging agent.

Embodiment 227: The immunoconjugate of embodiment 226, wherein the drugcomprises a drug selected from the group consisting of a calicheamicin,a duocarmycin, and a pyrrolobenzodiazepines.

Embodiment 228: The immunoconjugate of embodiment 227, wherein the drugcomprises a calicheamicin or a calicheamicin analog.

Embodiment 229: The immunoconjugate of embodiment 227, wherein the drugcomprises a duocarmycin.

Embodiment 230: The immunoconjugate of embodiment 229, wherein the drugcomprises a duocarmycin, selected from the group consisting ofduocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1,duocarmycin C2, duocarmycin D, duocarmycin SA, Cyclopropylbenzoindoleduocarmycin (CC-1065), Centanamycin, Rachelmycin, Adozelesin, Bizelesin,and Carzelesin.

Embodiment 231: The immunoconjugate of embodiment 227, wherein the drugcomprises a pyrrolobenzodiazepine or a pyrrolobenzodiazepine dimer.

Embodiment 232: The immunoconjugate of embodiment 231, wherein the drugcomprise a drug selected from the group consisting of Anthramycin (anddimers thereof), Mazethramycin (and dimers thereof), Tomaymycin (anddimers thereof), Prothracarcin (and dimers thereof), Chicamycin (anddimers thereof), Neothramycin A (and dimers thereof), Neothramycin B(and dimers thereof), DC-81 (and dimers thereof), Sibiromycin (anddimers thereof), Porothramycin A (and dimers thereof), Porothramycin B(and dimers thereof), Sibanomycin (and dimers thereof), Abbeymycin (anddimers thereof), SG2000, and SG2285.

Embodiment 233: The immunoconjugate of embodiment 221, wherein the drugcomprises a polymerase inhibitor.

Embodiment 234: The immunoconjugate of embodiment 233, wherein said drugcomprise a poly(ADP-ribose) polymerase (PARP) inhibitor.

Embodiment 235: The immunoconjugate of embodiment 234, wherein said drugcomprise a poly(ADP-ribose) polymerase (PARP) inhibitor selected fromthe group consisting of Iniparib (BSI 201), Talazoparib (BMN-673),Olaparib (AZD-2281), Olaparib, Rucaparib (AG014699, PF-01367338),Veliparib (ABT-888), CEP 9722, MK 4827, BGB-290, and 3-aminobenzamide.

Embodiment 236: The immunoconjugate according to any one of embodiments218-219, wherein said drug is selected from the group consisting ofauristatin, dolastatin, colchicine, combretastatin, and mTOR/PI3Kinhibitors.

Embodiment 237: The immunoconjugate according to any one of embodiments218-219, wherein said drug is selected from the group consisting offlourouracil (5-FU), capecitabine, 5-trifluoromethyl-2′-deoxyuridine,methotrexate sodium, raltitrexed, pemetrexed, cytosine Arabinoside,6-mercaptopurine, azathioprine, 6-thioguanine (6-TG), pentostatin,fludarabine phosphate, cladribine, floxuridine (5-fluoro-2),ribonucleotide reductase inhibitor (RNR), cyclophosphamide, neosar,ifosfamide, thiotepa, 1,3-bis(2-chloroethyl)-1-nitosourea (BCNU),1,-(2-chloroethyl)-3-cyclohexyl-lnitrosourea, methyl (CCNU),hexamethylmelamine, busulfan, procarbazine HCL, dacarbazine (DTIC),chlorambucil, melphalan, cisplatin, carboplatin, oxaliplatin,bendamustine, carmustine, chloromethine, dacarbazine (DTIC),fotemustine, lomustine, mannosulfan, nedaplatin, nimustine,prednimustine, ranimustine, satraplatin, semustine, streptozocin,temozolomide, treosulfan, triaziquone, triethylene melamine, thioTEPA,triplatin tetranitrate, trofosfamide, uramustine, doxorubicin,daunorubicin citrate, mitoxantrone, actinomycin D, etoposide, topotecanHCL, teniposide (VM-26), irinotecan HCL (CPT-11), camptothecin,belotecan, rubitecan, vincristine, vinblastine sulfate, vinorelbinetartrate, vindesine sulphate, paclitaxel, docetaxel, nanoparticlepaclitaxel, abraxane, ixabepilone, larotaxel, ortataxel, tesetaxel, andvinflunine.

Embodiment 238: The immunoconjugate according to any one of embodiments218-219, wherein said drug is selected from the group consisting ofcarboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin,erlotinib, etoposide, gemcitabine, imatinib mesylate, irinotecan,methotrexate, sorafinib, sunitinib, topotecan, vinblastine, andvincristine.

Embodiment 239: The immunoconjugate according to any one of embodiments218-219, wherein said drug is selected from the group consisting ofretinoic acid, a retinoic acid derivative, doxirubicin, vinblastine,vincristine, cyclophosphamide, ifosfamide, cisplatin, 5-fluorouracil, acamptothecin derivative, interferon, tamoxifen, and taxol. In certainembodiments the anti-cancer compound is selected from the groupconsisting of abraxane, doxorubicin, pamidronate disodium, anastrozole,exemestane, cyclophosphamide, epirubicin, toremifene, letrozole,trastuzumab, megestroltamoxifen, paclitaxel, docetaxel, capecitabine,goserelin acetate, and zoledronic acid.

Embodiment 240: The immunoconjugate of embodiment 215, wherein saidantibody is attached to a chelate including an isotope selected from thegroup consisting of ⁹⁹Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ¹¹³In, ⁹⁷Ru,⁶²Cu, ⁶⁴¹Cu, ⁵²Fe, ⁵²Mn, ⁵¹Cr, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er,¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm,¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh,and ¹¹¹Ag.

Embodiment 241: The immunoconjugate of embodiment 215, wherein saidantibody is attached to an alpha emitter.

Embodiment 242: The immunoconjugate of embodiment 241, wherein saidalpha emitter is bismuth 213.

Embodiment 243: The immunoconjugate of embodiment 215, wherein saidantibody is attached to a lipid or a liposome complexed with orcontaining an anti-cancer drug.

Embodiment 244: The immunoconjugate of embodiment 215, wherein saidantibody is attached to a detectable label.

Embodiment 245: The immunoconjugate of embodiment 244, wherein saidantibody is attached to a detectable label selected from the groupconsisting of a radioactive label, a radioopaque label, an MRI label,and a PET label.

Embodiment 246: A pharmaceutical formulation said formulation including:a pharmaceutically acceptable excipient and an antibody according to anyone of embodiments 1-214; and/or a pharmaceutically acceptable excipientand an immunoconjugate according to any one of embodiments 215-245.

Embodiment 247: The pharmaceutical formulation of embodiment 246,wherein said formulation is a unit dosage formulation.

Embodiment 248: The formulation according to any one of embodiments246-247, wherein said formulation is formulated for administration via aroute selected from the group consisting of oral administration, nasaladministration, rectal administration, intraperitoneal injection,intravascular injection, subcutaneous injection, transcutaneousadministration, and intramuscular injection.

Embodiment 249: A method of inhibiting the growth and/or proliferationof a cell that expresses or overexpresses CD46, said method including:contacting said cancer cell with an antibody according to any one ofembodiments 1-214; and/or contacting said cancer cell with animmunoconjugate including an antibody according to any one ofembodiments 1-214 attached to an effector that has cytostatic and/orcytotoxic activity.

Embodiment 250: The method of embodiment 249, wherein said methodcomprises contacting said cancer cell with an antibody according to anyone of embodiments 1-214.

Embodiment 251: The method of embodiment 249, wherein said methodcomprises contacting said cancer cell with an immunoconjugate includingan antibody according to any one of embodiments 1-214 attached to aneffector that has cytostatic and/or cytotoxic activity.

Embodiment 252: The method of embodiments 249-251, wherein said cell isa cancer cell.

Embodiment 253: The method of embodiment 252, wherein said cell is acancer cell that overexpresses CD46.

Embodiment 254: The method of embodiment 252, wherein said cancer cellis selected from the group consisting of ovarian cancer, colorectalcancer, breast cancer, lung cancer, prostate cancer, kidney cancer,pancreatic cancer, mesothelioma, lymphoma, liver cancer, urothelialcancer, stomach cancer, multiple myeloma, glioblastoma multiforme,glioma, neuroblastoma, and cervical cancer.

Embodiment 255: The method of embodiment 252, wherein said cancer cellis a prostate cancer cell.

Embodiment 256: The method of embodiment 255, wherein said cancer cellis a cell of a castration-resistant prostate cancer.

Embodiment 257: The method of embodiment 252, wherein said cancer cellis a cell of a multiple myeloma.

Embodiment 258: The method according to any one of embodiments 252-257,wherein said cell is a metastatic cell.

Embodiment 259: The method of embodiment 258, wherein said metastaticcell is a bone metastasis, a liver metastasis, a bladder metastasis,and/or a lymph node metastasis.

Embodiment 260: The method according to any one of embodiments 252-258,wherein said cell is a solid tumor cell.

Embodiment 261: The method according to any one of embodiments 249, and251-260, wherein said effector comprises a radionuclide and/or acytostatic drug.

Embodiment 262: The method of embodiment 261, wherein said effectorcomprises one or more of the following: a cytotoxic and/or cytostaticdrug; a lipid or liposome containing a cytotoxic and/or cytostatic drug;a polymeric drug carrier including a cytotoxic and/or cytostatic drug;and a nanoparticle drug carrier including a cytotoxic and/or cytostaticdrug.

Embodiment 263: The method of embodiment 262, wherein said drug is ananti-cancer drug.

Embodiment 264: The method of embodiment 263, wherein said drug isselected from the group consisting of auristatin, dolastatin,colchicine, combretastatin, and mTOR/PI3K inhibitors.

Embodiment 265: The method of embodiment 263, wherein said drug ismonomethyl auristatin F.

Embodiment 266: The method of embodiment 263, wherein said drug isselected from the group consisting of flourouracil (5-FU), capecitabine,5-trifluoromethyl-2′-deoxyuridine, methotrexate sodium, raltitrexed,pemetrexed, cytosine Arabinoside, 6-mercaptopurine, azathioprine,6-thioguanine (6-TG), pentostatin, fludarabine phosphate, cladribine,floxuridine (5-fluoro-2), ribonucleotide reductase inhibitor (RNR),cyclophosphamide, neosar, ifosfamide, thiotepa,1,3-bis(2-chloroethyl)-1-nitosourea (BCNU),1,-(2-chloroethyl)-3-cyclohexyl-lnitrosourea, methyl (CCNU),hexamethylmelamine, busulfan, procarbazine HCL, dacarbazine (DTIC),chlorambucil, melphalan, cisplatin, carboplatin, oxaliplatin,bendamustine, carmustine, chloromethine, dacarbazine (DTIC),fotemustine, lomustine, mannosulfan, nedaplatin, nimustine,prednimustine, ranimustine, satraplatin, semustine, streptozocin,temozolomide, treosulfan, triaziquone, triethylene melamine, thioTEPA,triplatin tetranitrate, trofosfamide, uramustine, doxorubicin,daunorubicin citrate, mitoxantrone, actinomycin D, etoposide, topotecanHCL, teniposide (VM-26), irinotecan HCL (CPT-11), camptothecin,belotecan, rubitecan, vincristine, vinblastine sulfate, vinorelbinetartrate, vindesine sulphate, paclitaxel, docetaxel, nanoparticlepaclitaxel, abraxane, ixabepilone, larotaxel, ortataxel, tesetaxel, andvinflunine.

Embodiment 267: The method of embodiment 263, wherein said drug isselected from the group consisting of carboplatin, cisplatin,cyclophosphamide, docetaxel, doxorubicin, erlotinib, etoposide,gemcitabine, imatinib mesylate, irinotecan, methotrexate, sorafinib,sunitinib, topotecan, vinblastine, and vincristine.

Embodiment 268: The method of embodiment 263, wherein said drug isselected from the group consisting of retinoic acid, a retinoic acidderivative, doxirubicin, vinblastine, vincristine, cyclophosphamide,ifosfamide, cisplatin, 5-fluorouracil, a camptothecin derivative,interferon, tamoxifen, and taxol. In certain embodiments the anti-cancercompound is selected from the group consisting of abraxane, doxorubicin,pamidronate disodium, anastrozole, exemestane, cyclophosphamide,epirubicin, toremifene, letrozole, trastuzumab, megestroltamoxifen,paclitaxel, docetaxel, capecitabine, goserelin acetate, and zoledronicacid.

Embodiment 269: The method according to any one of embodiments 262-268,wherein said drug is conjugated to said antibody.

Embodiment 270: The method according to any one of embodiments 262-268,wherein said drug is contained in a lipid or liposome attached to saidantibody.

Embodiment 271: The method according to any one of embodiments 262-268,wherein said drug is contained in a polymeric and/or nanoparticlecarrier attached to said antibody.

Embodiment 272: The method of embodiment 249, and 251-260, wherein saideffector comprises a cytotoxin.

Embodiment 273: The method of embodiment 272, wherein said cytotoxin isselected from the group consisting of Diphtheria toxin, Pseudomonasexotoxin, ricin, abrin, saporin, and thymidine kinase.

Embodiment 274: The method of embodiment 249, wherein said effectorcomprises a radionuclide.

Embodiment 275: The method according to any one of embodiments 249-274,wherein said immunoconjugate or antibody is administered in apharmaceutical composition including a pharmaceutical acceptablecarrier.

Embodiment 276: The method according to any one of embodiments 249-275,wherein said administering comprises administering to a human.

Embodiment 277: The method according to any one of embodiments 249-275,wherein said administering comprises administering to a non-humanmammal.

Embodiment 278: The method according to any one of embodiments 249-277,wherein said administering comprises administering parenterally.

Embodiment 279: The method according to any one of embodiments 249-277,wherein said administering comprises administering into a tumor or asurgical site.

Embodiment 280: The method according to any one of embodiments 249-279,wherein said immunoconjugate is administered as an adjunct therapy tosurgery and/or radiotherapy.

Embodiment 281: The method according to any one of embodiments 249-279,wherein said antibody and/or immunoconjugate is administered inconjunction with another anti-cancer drug and/or a hormone.

Embodiment 282: The method of embodiment 281, wherein said antibodyand/or immunoconjugate is administered in conjunction with abirateroneand/or enzalutamide.

Embodiment 283: The method of embodiment 282, wherein said cellscomprise prostate cancer cells.

Embodiment 284: The method of embodiment 283, wherein said prostatecancer cells comprise neuroendocrine prostate cancer (NEPC) cells.

Embodiment 285: The method of embodiment 283, wherein said prostatecancer cells comprise metastatic castration resistant prostate cancer(mCRPC) cells resistant to abiraterone (Abi) or enzalutamide (Enz).

Embodiment 286: A method of detecting a cancer cell of a cancer thatexpresses or overexpresses CD46, said method including: contacting saidcancer cell with a immunoconjugate including an antibody according toany one of embodiments 1-214 attached to a detectable label; anddetecting the presence and/or location of said detectable label wherethe presence and/or location is an indicator of the location and/orpresence of a cancer cell.

Embodiment 287: The method of embodiment 286, wherein said labelcomprises a label selected from the group consisting of a radioactivelabel, a radioopaque label, an MRI label, a PET label, and an SPECTlabel.

Embodiment 288: The method of embodiment 286, wherein said detectablelabel is selected from the group consisting of a gamma-emitter, apositron-emitter, an x-ray emitter, an alpha emitter, and afluorescence-emitter.

Embodiment 289: The method according to any one of embodiments 286-288,wherein said cancer cell is selected from the group consisting ofovarian cancer, colorectal cancer, breast cancer, lung cancer, prostatecancer, kidney cancer, pancreatic cancer, mesothelioma, lymphoma, livercancer, urothelial cancer, stomach cancer, multiple myeloma, glioma,neuroblastoma, and cervical cancer.

Embodiment 290: The method of embodiment 289, wherein said cancer cellis a prostate cancer cell.

Embodiment 291: The method of embodiment 290, wherein said cancer cellis a cell of a castration-resistant prostate cancer.

Embodiment 292: The method of embodiment 289, wherein said cancer cellis a cell of a multiple myeloma.

Embodiment 293: The method according to any one of embodiments 286-292,wherein said contacting comprises administering said immunoconjugate toa non-human mammal.

Embodiment 294: The method according to any one of embodiments 286-292,wherein said contacting comprises administering said immunoconjugate toa human.

Embodiment 295: The method according to any one of embodiments 286-294,wherein said detecting comprises detecting said label in vivo.

Embodiment 296: The method of embodiment 295, wherein said detectingcomprises using a detection method selected from the group consisting ofX-ray, PET, SPECT, MRI, and CAT.

Embodiment 297: The method according to any one of embodiments 286-294,wherein said detecting comprises detecting said label ex vivo in abiopsy or a sample derived from a biopsy.

Embodiment 298: A nucleic acid encoding an antibody or a fragment of anantibody according to any of embodiments 1-214.

Embodiment 299: An expression vector including the nucleic acid ofembodiment 298.

Embodiment 300: A cell including the expression vector of embodiment299.

Definitions

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The term also includes variants on the traditional peptidelinkage joining the amino acids making up the polypeptide.

The terms “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein refer to at least two nucleotides covalently linked together. Anucleic acid of the present invention is preferably single-stranded ordouble stranded and will generally contain phosphodiester bonds,although in some cases, as outlined below, nucleic acid analogs areincluded that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925) andreferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl etal. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. AcidsRes. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al.(1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) ChemicaScripta 26: 1419), phosphorothioate (Mag et al. (1991) Nucleic AcidsRes. 19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu etal. (1989) J. Am. Chem. Soc. 111:2321, O-methylphophoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al.(1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566;Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acidsinclude those with positive backbones (Denpcy et al. (1995) Proc. Natl.Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl.Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470;Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and3, ASC Symposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994),Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J.Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Ed. Y. S. Sanghui and P. Dan Cook.Nucleic acids containing one or more carbocyclic sugars are alsoincluded within the definition of nucleic acids (see Jenkins et al.(1995), Chem. Soc. Rev. pp 169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. These modificationsof the ribose-phosphate backbone may be done to facilitate the additionof additional moieties such as labels, or to increase the stability andhalf-life of such molecules in physiological environments.

The term “residue” as used herein refers to natural, synthetic, ormodified amino acids.

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H)1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into aFab′ monomer. The Fab′ monomer is essentially a Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Certain preferred antibodies includesingle chain antibodies (antibodies that exist as a single polypeptidechain), more preferably single chain Fv antibodies (sFv or scFv) inwhich a variable heavy and a variable light chain are joined together(directly or through a peptide linker) to form a continuous polypeptide.The single chain Fv antibody is a covalently linked V_(H-)V_(L)heterodimer which may be expressed from a nucleic acid including V_(H)-and V_(L)-encoding sequences either joined directly or joined by apeptide-encoding linker. Huston, et al. (1988) Proc. Nat. Acad. Sci.USA, 85: 5879-5883. While the V_(H) and V_(L) are connected to each as asingle polypeptide chain, the V_(H) and V_(L) domains associatenon-covalently. The first functional antibody molecules to be expressedon the surface of filamentous phage were single-chain Fv's (scFv),however, alternative expression strategies have also been successful.For example Fab molecules can be displayed on phage if one of the chains(heavy or light) is fused to g3 capsid protein and the complementarychain exported to the periplasm as a soluble molecule. The two chainscan be encoded on the same or on different replicons; the importantpoint is that the two antibody chains in each Fab molecule assemblepost-translationally and the dimer is incorporated into the phageparticle via linkage of one of the chains to, e.g., g3p (see, e.g., U.S.Pat. No. 5,733,743).

The scFv antibodies and a number of other structures converting thenaturally aggregated, but chemically separated light and heavypolypeptide chains from an antibody V region into a molecule that foldsinto a three dimensional structure substantially similar to thestructure of an antigen-binding site are known to those of skill in theart (see e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778).Particularly preferred antibodies should include all that have beendisplayed on phage (e.g., scFv, Fv, Fab and disulfide linked Fv (Reiteret al. (1995) Protein Eng. 8: 1323-1331).

The term “specifically binds”, as used herein, when referring to abiomolecule (e.g., protein, nucleic acid, antibody, etc.), refers to abinding reaction that is determinative of the presence biomolecule inheterogeneous population of molecules (e.g., proteins and otherbiologics). Thus, under designated conditions (e.g. immunoassayconditions in the case of an antibody or stringent hybridizationconditions in the case of a nucleic acid), the specified ligand orantibody binds to its particular “target” molecule and does not bind ina significant amount to other molecules present in the sample.

The phrase “inhibition of proliferation of a cell expressing CD46” asused herein, refers to the ability of an anti-CD46 antibody orimmunoconjugate described herein decrease, preferably to statisticallysignificantly decrease proliferation of a cell expressing CD46 relativeto the proliferation in the absence of the antibody or immunoconjugate.In one embodiment, the proliferation of a cell expressing CD46 (e.g., acancer cell) may be decreased by at least 10%, or at least 20%, or atleast 30%, or at least 40%, or at least 50%, or at least 60%, or atleast 70%, or at least 80%, or at least 90%, or 100% when the cells arecontacted with the antibody or antigen binding portion thereof or animmunoconjugate described herein, relative to the proliferation measuredin the absence of the antibody or antigen binding portion thereof orimmunoconjugate (control). Cellular proliferation can be assayed usingart recognized techniques which measure rate of cell division, thefraction of cells within a cell population undergoing cell division,and/or rate of cell loss from a cell population due to terminaldifferentiation or cell death (e.g., using a cell titer glow assay orthymidine incorporation).

The phrase “inhibition of the migration of cells expressing CD46” asused herein, refers to the ability of an anti-CD46 antibody or anantigen-binding portion thereof or an immunoconjugate described hereinto decrease, preferably to statistically significantly decrease themigration of a cell expressing CD46 relative to the migration of thecell in the absence of the antibody. In one embodiment, the migration ofa cell expressing CD46 (e.g., a cancer cell) may be decreased by atleast 10%, or at least 20%, or at least 30%, or at least 40%, or atleast 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 90%, or 100% when the cells are contacted with the antibody orantigen binding portion thereof or immunoconjugate thereof, relative tocell migration measured in the absence of the antibody or antigenbinding portion thereof or immunoconjugate thereof (control). Cellmigration can be assayed using art recognized techniques. In variousembodiments, it is contemplated that the antibodies and/or theimmunoconjugates thereof described herein can inhibit the migration ofcells (e.g., cancer cells as described herein) expressing oroverexpressing CD46.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., CD46 domain 1 and/or domain 2). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), CL andCH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; (iii) aFd fragment consisting of the V_(H) and CH1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb including VH and VL domains; (vi) a dAb fragment(see, e.g., Ward et al. (1989) Nature 341: 544-546), which consists of aV_(H) domain; (vii) a dAb which consists of a V_(H) or a V_(L) domain;and (viii) an isolated complementarity determining region (CDR) or (ix)a combination of two or more isolated CDRs which may optionally bejoined by a synthetic linker. Furthermore, although the two domains ofthe Fv fragment, V_(L) and V_(H), can be coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which theV_(L) and V-regions pair to form monovalent molecules (known as singlechain Fv (scFv); see e.g., Bird et al. (1988) Science 242: 423-426; andHuston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Suchsingle chain antibodies are also intended to be encompassed within theterm “antigen-binding portion” of an antibody. These antibody fragmentsare obtained using conventional techniques known to those with skill inthe art, and the fragments are screened for utility in the same manneras are intact antibodies. Antigen-binding portions can be produced byrecombinant DNA techniques, or by enzymatic or chemical cleavage ofintact immunoglobulins.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. Monoclonal antibodies can be prepared using any art recognizedtechnique and those described herein such as, for example, a hybridomamethod, as described by Kohler et al. (1975) Nature, 256: 495, atransgenic animal, as described by, for example, (see e.g., Lonberg, etal. (1994) Nature 368(6474): 856-859), recombinant DNA methods (see,e.g., U.S. Pat. No. 4,816,567), or using phage antibody libraries usingthe techniques described in, for example, Clackson et al. (1991) Nature,352: 624-628, and Marks et al. (1991) J. Mol. Biol., 222: 581-597.Monoclonal antibodies include chimeric antibodies, human antibodies andhumanized antibodies and may occur naturally or be recombinantlyproduced.

The term “recombinant antibody,” refers to antibodies that are prepared,expressed, created or isolated by recombinant means, such as (a)antibodies isolated from an animal (e.g., a mouse) that is transgenic ortranschromosomal for immunoglobulin genes (e.g., human immunoglobulingenes) or a hybridoma prepared therefrom, (b) antibodies isolated from ahost cell transformed to express the antibody, e.g., from atransfectoma, (c) antibodies isolated from a recombinant, combinatorialantibody library (e.g., containing human antibody sequences) using phagedisplay, and (d) antibodies prepared, expressed, created or isolated byany other means that involve splicing of immunoglobulin gene sequences(e.g., human immunoglobulin genes) to other DNA sequences. Suchrecombinant antibodies may have variable and constant regions derivedfrom human germline immunoglobulin sequences. In certain embodiments,however, such recombinant human antibodies can be subjected to in vitromutagenesis and thus the amino acid sequences of the V_(H) and V_(L)regions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline V- and V_(L) sequences, may notnaturally exist within the human antibody germline repertoire in vivo.

The term “chimeric immunoglobulin” or antibody refers to animmunoglobulin or antibody whose variable regions derive from a firstspecies and whose constant regions derive from a second species.Chimeric immunoglobulins or antibodies can be constructed, for exampleby genetic engineering, from immunoglobulin gene segments belonging todifferent species.

The term “human antibody,” as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences asdescribed, for example, by Kabat et al. (See Kabat, et al. (1991)Sequences of proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies may include amino acid residues not encoded by humangermline immunoglobulin sequences (e.g., mutations introduced by randomor site-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The human antibody can have at least one or more amino acids replacedwith an amino acid residue, e.g., an activity enhancing amino acidresidue which is not encoded by the human germline immunoglobulinsequence. Typically, the human antibody can have up to twenty positionsreplaced with amino acid residues which are not part of the humangermline immunoglobulin sequence. In a particular embodiment, thesereplacements are within the CDR regions as described in detail below.

The term “humanized immunoglobulin” or “humanized antibody” refers to animmunoglobulin or antibody that includes at least one humanizedimmunoglobulin or antibody chain (i.e., at least one humanized light orheavy chain). The term “humanized immunoglobulin chain” or “humanizedantibody chain” (i.e., a “humanized immunoglobulin light chain” or“humanized immunoglobulin heavy chain”) refers to an immunoglobulin orantibody chain (i.e., a light or heavy chain, respectively) having avariable region that includes a variable framework region substantiallyfrom a human immunoglobulin or antibody and complementarity determiningregions (CDRs) (e.g., at least one CDR, preferably two CDRs, morepreferably three CDRs) substantially from a non-human immunoglobulin orantibody, and further includes constant regions (e.g., at least oneconstant region or portion thereof, in the case of a light chain, andpreferably three constant regions in the case of a heavy chain). Theterm “humanized variable region” (e.g., “humanized light chain variableregion” or “humanized heavy chain variable region”) refers to a variableregion that includes a variable framework region substantially from ahuman immunoglobulin or antibody and complementarity determining regions(CDRs) substantially from a non-human immunoglobulin or antibody.

As used herein, a “heterologous antibody” is defined in relation to thetransgenic non-human organism or plant producing such an antibody.

An “isolated antibody,” as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds to CD46 is substantially free of antibodies that specifically bindantigens other than CD46). In addition, an isolated antibody istypically substantially free of other cellular material and/orchemicals. In one embodiment, a combination of “isolated” monoclonalantibodies having different CD46 binding specificities are combined in awell defined composition.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes. In oneembodiment, an antibody or antigen binding portion thereof is of anisotype selected from an IgG1, an IgG2, an IgG3, an IgG4, an IgM, anIgA1, an IgA2, an IgAsec, an IgD, or an IgE antibody isotype. In someembodiments, a monoclonal antibody of the invention is of the IgG1isotype. In other embodiments, a monoclonal antibody of the invention isof the IgG2 isotype.

An “antigen” is an entity (e.g., a proteinaceous entity or peptide) towhich an antibody or antigen-binding portion thereof binds. In variousembodiments of the present invention, an antigen is CD46, e.g., aspresented on a cell (e.g., a CD46 positive cancer cell).

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody specifically binds.Epitopes can be formed both from contiguous amino acids or noncontiguousamino acids juxtaposed by tertiary folding of a protein. Epitopes formedfrom contiguous amino acids are typically retained on exposure todenaturing solvents, whereas epitopes formed by tertiary folding aretypically lost on treatment with denaturing solvents. An epitopetypically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15 amino acids in a unique spatial conformation. Methods of determiningspatial conformation of epitopes include techniques in the art and thosedescribed herein, for example, x-ray crystallography and 2-dimensionalnuclear magnetic resonance (see, e.g., Epitope Mapping Protocols inMethods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

Also contemplated herein are antibodies that bind the same or anoverlapping epitope as the YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7,SB2, 2C8, and/or UA8kappa antibodies described herein. Antibodies thatrecognize the same epitope can be identified using routine techniquessuch as an immunoassay, for example, by showing the ability of oneantibody to block the binding of another antibody to a target antigen,i.e., a competitive binding assay. Competitive binding is determined inan assay in which the immunoglobulin under test inhibits specificbinding of a reference antibody to a common antigen, such as CD46 domain1 and/or domain 2. Numerous types of competitive binding assays areknown, for example: solid phase direct or indirect radioimmunoassay(RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwichcompetition assay (see, e.g., Stahli et al. (1983) Meth. Enzymol., 9:242); solid phase direct biotin-avidin EIA (see Kirkland et al., (1986)J. Immunol. 137: 3614); solid phase direct labeled assay, solid phasedirect labeled sandwich assay (see, e.g., Harlow and Lane (1988)Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid phasedirect label RIA using, e.g., ¹²⁵I label (see, e.g., Morel et al.,(1988) Mol. Immunol. 25(1): 7); solid phase direct biotin-avidin EIA(Cheung et al. (1990) Virology 176: 546); and direct labeled RIA.(Moldenhauer et al. (1990) Scand J. Immunol. 32: 77). Typically, such anassay involves the use of purified antigen (e.g., CD46 domain 1 and/ordomain 2) bound to a solid surface or cells bearing either of these, anunlabeled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Usually, when a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to a common antigen by at least50-55%, 55-60%, 60-65%, 65-70% 70-75% or more.

As used herein, the terms “specific binding,” “specifically binds,”“selective binding,” and “selectively binds,” mean that an antibody orantigen-binding portion thereof, exhibits appreciable affinity for aparticular antigen or epitope and, generally, does not exhibitsignificant cross-reactivity with other antigens and epitopes.“Appreciable” or preferred binding includes binding with an affinity ofat least (KD equal to or less than) 10⁻⁶ M, 10⁻⁷M, 10⁻⁸M, 10⁻⁹M, 10⁻¹⁰M, or 10⁻¹¹ M. Affinities greater than 10⁻⁹ M, preferably greater than10⁻¹⁰ M are more preferred. Values intermediate of those set forthherein are also intended to be within the scope of the present inventionand a preferred binding affinity can be indicated as a range ofaffinities, for example, 10⁻⁶ M to 10⁻¹¹ M, preferably 10⁻⁷ M or 10⁻⁸ Mto 10⁻¹⁰ M. An antibody that “does not exhibit significantcross-reactivity” is one that will not appreciably bind to anundesirable entity (e.g., an undesirable proteinaceous entity). Forexample, in one embodiment, an antibody or antigen-binding portionthereof that specifically binds to CD46 (e.g., domain 1 and/or domain 2)protein but will not significantly react with other molecules andnon-CD46 proteins or peptides. Specific or selective binding can bedetermined according to any art-recognized means for determining suchbinding, including, for example, according to Scatchard analysis and/orcompetitive binding assays.

The term “K_(D),” as used herein, is intended to refer to thedissociation equilibrium constant of a particular antibody-antigeninteraction or the affinity of an antibody for an antigen. In oneembodiment, the antibody or antigen binding portion thereof according tothe present invention binds an antigen (e.g., CD46 domain 1 and/ordomain 2) with an affinity (K_(D)) of 5 nM or better (i.e., or less)(e.g., 40 nM or 30 nM or 20 nM or 10 nM or less), as measured using asurface plasmon resonance assay or a cell binding assay. In a particularembodiment, an antibody or antigen binding portion thereof according tothe present invention binds CD46 with an affinity (K_(D)) of 5 nM orbetter (e.g., 4 nM, 2 nM, 1.5 nM, 1.4 nM, 1.3 nM, 1 nM or less), asmeasured by a surface plasmon resonance assay or a cell binding assay.In other embodiments, an antibody or antigen binding portion thereofbinds an antigen (e.g., CD46) with an affinity (K_(D)) of approximatelyless than 10⁻¹⁰ M, or 100×10⁻¹¹ M, or 10×10⁻¹¹ M, or even lower usinglive prostate tumor cells by FACS.

The term “K_(off),” as used herein, is intended to refer to the off rateconstant for the dissociation of an antibody from the antibody/antigencomplex.

The term “EC50,” as used herein, refers to the concentration of anantibody or an antigen-binding portion thereof or an immunoconjugatedescribed herein, that induces a response, either in an in vitro or anin vivo assay, which is 50% of the maximal response, i.e., halfwaybetween the maximal response and the baseline.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

The term “modifying,” or “modification,” as used herein, is intended torefer to changing one or more amino acids in the antibodies orantigen-binding portions thereof. The change can be produced by adding,substituting or deleting an amino acid at one or more positions. Thechange can be produced using known techniques, such as PCR mutagenesis.For example, in some embodiments, an antibody or an antigen-bindingportion thereof identified using the methods of the invention can bemodified, to thereby modify the binding affinity of the antibody orantigen-binding portion thereof to CD46.

In certain embodiments “conservative amino acid substitutions” in thesequences of the anti-CD46 antibodies described herein, i.e., nucleotideand amino acid sequence modifications that do not abrogate the bindingof the antibody encoded by the nucleotide sequence or containing theamino acid sequence, to the antigen, e.g., CD46 are contemplated.Conservative amino acid substitutions include the substitution of anamino acid in one class by an amino acid of the same class, where aclass is defined by common physicochemical amino acid side chainproperties and high substitution frequencies in homologous proteinsfound in nature, as determined, for example, by a standard Dayhofffrequency exchange matrix or BLOSUM matrix. Six general classes of aminoacid side chains have been categorized and include: Class I (Cys); ClassII (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV(His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr,Trp). For example, substitution of an Asp for another class III residuesuch as Asn, Gln, or Glu, is a conservative substitution. Thus, apredicted nonessential amino acid residue in an anti-CD46 antibody ispreferably replaced with another amino acid residue from the same class.Methods of identifying nucleotide and amino acid conservativesubstitutions that do not eliminate antigen binding are well-known inthe art (see, e.g., Brummell et al. (1993) Biochem. 32: 1180-1187;Kobayashi et al. (1999) Protein Eng. 12(10): 879-884; and Burks et al.(1997) Proc. Natl. Acad. Sci. USA 94: 412-417).

The term “non-conservative amino acid substitution” refers to thesubstitution of an amino acid in one class with an amino acid fromanother class; for example, substitution of an Ala, a class II residue,with a class III residue such as Asp, Asn, Glu, or Gln.

In another embodiment, mutations (conservative or non-conservative) canbe introduced randomly along all or part of an anti-CD46 antibody codingsequence, such as by saturation mutagenesis, and the resulting modifiedantibodies can be screened for binding activity.

A “consensus sequence” is a sequence formed from the most frequentlyoccurring amino acids (or nucleotides) in a family of related sequences(See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft,Weinheim, Germany 1987). In a family of proteins, each position in theconsensus sequence is occupied by the amino acid occurring mostfrequently at that position in the family. If two amino acids occurequally frequently, either can be included in the consensus sequence. A“consensus framework” of an immunoglobulin refers to a framework regionin the consensus immunoglobulin sequence.

Similarly, the consensus sequence for the CDRs of can be derived byoptimal alignment of the CDR amino acid sequences of anti-CD46antibodies described herein.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions.times.100), taking into accountthe number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences. The comparison ofsequences and determination of percent identity between two sequencescan be accomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software, using a NWSgapdna.CMP matrixand a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2,3, 4, 5, or 6. The percent identity between two nucleotide or amino acidsequences can also be determined using the algorithm of Meyers andMiller (1989) CABIOS, 4: 11-17, which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4. In addition, the percentidentity between two amino acid sequences can be determined using theNeedleman and Wunsch (1970) J. Mol. Biol. 48: 444-453 algorithm whichhas been incorporated into the GAP program in the GCG software package,using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the contemplated herein canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.

25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used.

The nucleic acid compositions described herein (e.g., nucleic acidsencoding all or a portion of an anti-CD46 antibody or immunoconjugate)while often in a native sequence (except for modified restriction sitesand the like), from either cDNA, genomic or mixtures thereof may bemutated, in accordance with standard techniques to provide variantsequences. For coding sequences, these mutations, may affect amino acidsequence as desired. In particular, DNA sequences substantiallyhomologous to or derived from native V, D, J, constant, switches andother such sequences described herein are contemplated (where “derived”indicates that a sequence is identical or modified from anothersequence).

The term “operably linked” refers to a nucleic acid sequence placed intoa functional relationship with another nucleic acid sequence. Forexample, DNA for a presequence or secretory leader is operably linked toDNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Generally, “operably linked” means that the DNA sequences being linkedare contiguous, and, in the case of a secretory leader, contiguous andin reading phase. However, enhancers do not have to be contiguous.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, the synthetic oligonucleotide adaptors orlinkers are used in accordance with conventional practice. A nucleicacid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. The terms, “plasmid” and “vector” may be usedinterchangeably. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), that serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which an expression vectorhas been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell but to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The terms “treat,” “treating,” and “treatment,” as used herein, refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration to a subject (e.g., a subject in needthereof), an anti-CD46 antibody or antigen binding portion or animmunoconjugate comprising such an antibody or antigen binding portiondescribed herein. In certain embodiments the subject is a subjectdiagnosed with and/or under treatment for a CD46 positive cancer (e.g.,prostate cancer) in order to prevent, cure, delay, reduce the severityof, or ameliorate one or more symptoms of the disease or disorder orrecurring disease or disorder, or in order to prolong the survival of asubject beyond that expected in the absence of such treatment.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A CD46 positive cancer refers to a cancercharacterized by cells that express or overexpress CD46. IllustrativeCD46 cancers include, but are not limited to, ovarian cancer, breastcancer, lung cancer, prostate cancer, colon cancer, kidney cancer, andpancreatic cancer.

The term “effective amount,” as used herein, refers to that amount of ananti-CD46 antibody or an antigen binding portion thereof and/or animmunoconjugate thereof, that is sufficient to effect treatment,prognosis or diagnosis of a disease associated with the growth and/orproliferation of CD46 positive cells (e.g., a CD46 positive cancer), asdescribed herein, when administered to a subject. A therapeuticallyeffective amount will vary depending upon the subject and diseasecondition being treated, the weight and age of the subject, the severityof the disease condition, the manner of administration and the like,which can readily be determined by one of ordinary skill in the art. Thedosages for administration can range from, for example, about 1 ng toabout 10,000 mg, about 5 ng to about 9,500 mg, about 10 ng to about9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about 7,500 mg,about 40 ng to about 7,000 mg, about 50 ng to about 6,500 mg, about 100ng to about 6,000 mg, about 200 ng to about 5,500 mg, about 300 ng toabout 5,000 mg, about 400 ng to about 4,500 mg, about 500 ng to about4,000 mg, about 1 μg to about 3,500 mg, about 5 μg to about 3,000 mg,about 10 μg to about 2,600 mg, about 20 μg to about 2,575 mg, about 30μg to about 2,550 mg, about 40 μg to about 2,500 mg, about 50 μg toabout 2,475 mg, about 100 μg to about 2,450 mg, about 200 μg to about2,425 mg, about 300 μg to about 2,000, about 400 μg to about 1,175 mg,about 500 μg to about 1,150 mg, about 0.5 mg to about 1,125 mg, about 1mg to about 1,100 mg, about 1.25 mg to about 1,075 mg, about 1.5 mg toabout 1,050 mg, about 2.0 mg to about 1,025 mg, about 2.5 mg to about1,000 mg, about 3.0 mg to about 975 mg, about 3.5 mg to about 950 mg,about 4.0 mg to about 925 mg, about 4.5 mg to about 900 mg, about 5 mgto about 875 mg, about 10 mg to about 850 mg, about 20 mg to about 825mg, about 30 mg to about 800 mg, about 40 mg to about 775 mg, about 50mg to about 750 mg, about 100 mg to about 725 mg, about 200 mg to about700 mg, about 300 mg to about 675 mg, about 400 mg to about 650 mg,about 500 mg, or about 525 mg to about 625 mg, of an anti-CD46 antibodydescribed herein and/or antigen binding portion thereof, and/orimmunoconjugate thereof as described herein. Dosage regiments may beadjusted to provide the optimum therapeutic response. An effectiveamount is also one in which any toxic or detrimental effects (i.e., sideeffects) of an antibody or antigen binding portion thereof are minimizedand/or outweighed by the beneficial effects.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

As used herein, the term “subject” includes any human or non-humananimal. For example, the methods and compositions of the presentinvention can be used to treat a subject having cancer. In a particularembodiment, the subject is a human. The term “non-human animal” includesall vertebrates, e.g., mammals and non-mammals, such as non-humanprimates, sheep, dog, cow, chickens, amphibians, reptiles, etc.

An “effector” refers to any molecule or combination of molecules whoseactivity it is desired to deliver/into and/or localize at cell.Effectors include, but are not limited to labels, cytotoxins, enzymes,growth factors, transcription factors, antibodies, drugs, etc.

The phrase “inhibiting the growth and/or proliferation”, e.g. of cancercells includes inter alia inducing cellular apoptosis or other cellkilling mechanisms, reducing the invasiveness of the cells, stalling thecells at a point in the cell cycle, and the like.

The term “immunoconjugate” refers to an antibody attached to one or moreeffectors or to a plurality of antibodies attached to one or moreeffectors. The term “immunoconjugate” is intended to include effectorschemically conjugated to the antibodies as well as antibodies expressesas a fusion protein where the antibody (or a portion thereof) isdirectly attached or attached through a linker to a peptide effector orto an effector comprising a peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows VH framework and CDR regions for YS5 (SEQ ID NO:1), YS5F(SEQ ID NO:2), YS5v1D (SEQ ID NO:3), SB1HGNY (SEQ ID NO:4), YS12 (SEQ IDNO:5), 3G7RY (aka 3G8) (SEQ ID NO:6), YS6 (SEQ ID NO:7), YS1 (SEQ IDNO:8), YS3 (SEQ ID NO:9), YS4 (SEQ ID NO:10), YS8 (SEQ ID NO:11), YS7(SEQ ID NO:12), YS9 (SEQ ID NO:13), YS10 (SEQ ID NO:14), YS11 (SEQ IDNO:15), 3G7HY (SEQ ID NO:16), 3G7NY (SEQ ID NO:17), 3G7 (SEQ ID NO:18),SB2 (SEQ ID NO:19), 2C8 (SEQ ID NO:20), and UA8kappa (SEQ ID NO:21).FIG. 1B shows VL framework and CDR regions for YS5 (SEQ ID NO:22), YS5F(SEQ ID NO:23), YS5v1D (SEQ ID NO:24), SB1HGNY (SEQ ID NO:25), YS12 (SEQID NO:26), 3G7RY (aka3G8) (SEQ ID NO:27), YS6 (SEQ ID NO:28), YS1 (SEQID NO:29), YS3 (SEQ ID NO:30), YS4 (SEQ ID NO:31), YS8 (SEQ ID NO:32),YS7 (SEQ ID NO:33), YS9 (SEQ ID NO:34), YS10 (SEQ ID NO:35), YS11 (SEQID NO:36), 3G7HY (SEQ ID NO:37), 3G7NY (SEQ ID NO:38), 3G7 (SEQ IDNO:39), SB2 (SEQ ID NO:40), 2C8 (SEQ ID NO:41), and UA8kappa (SEQ IDNO:42).

FIG. 2. YS5 IgG1 K_(D) measurement on Du-145 cells. YS5 was incubatedwith Du-145 cells at 4° C. overnight and binding analyzed by FACS. Meanfluorescence intensity (MFI) values were curve-fit using Prism(GraphPad) to generate an estimated KD value of 2.19+/−0.73 nM.

FIG. 3. YS12 IgG1 K_(D) measurement on Du-145 cells. YS12 was incubatedwith Du-145 cells at 4° C. overnight and binding analyzed by FACS. MFIvalues were curve-fit using Prism (GraphPad) to generate an estimated KDvalue of 0.043+/−0.019 nM.

FIG. 4. Our anti-CD46 antibodies bind to both human and cynomolgusmonkey CD46. FACS analysis were performed on CHO cells transfected witheither human (left panel) and cyno monkey CD46 (right panel). The resultfor YS5 is shown but all our antibodies bind to both human andcynomolgus CD46. Ctr IgG: a non-binding human IgG1. Ctr: CHO stainedwith secondary antibodies only.

FIG. 5. YS5 K_(D) measurement on CHO cells transfected with human CD46.YS5 was incubated with CHO-huCD46 cells at 4° C. overnight and bindinganalyzed by FACS. MFI values were curve-fit using Prism (GraphPad) togenerate an estimated K_(D) value of 0.867+/−0.299 nM.

FIG. 6. YS5 K_(D) measurement on CHO cells transfected with cynomolgusmonkey CD46. YS5 was incubated with CHO-cynoCD46 cells at 4° C.overnight and binding analyzed by FACS. MFI values were curve-fit usingPrism (GraphPad) to generate an estimated KD value of 1.952+/−0.508 nM.

FIG. 7. Epitope mapping by competition assay. FACS binding on Du145cells with or without UA20Fc as a competitor. UA20Fc vs. UA20Fc servesas a control for complete competition. YS5, YS12 and 3G8 (i.e., 3G7 aka3G8) showed a different competition pattern than that of SB1HGNY, thusdefining two groups with non-overlapping epitopes.

FIG. 8. Competition with Edmonston strain measles virus H protein.Antibody binding to Du-145 cells in the presence or absence of excessrecombinant H protein-Fc fusion was measured by FACS. H protein-Fc vs. Hprotein-Fc was done as a positive control (total competition), againstwhich the MFI values were normalized to generate the normalizedcompetition index. A non-CD46 binding, Du-145 cell binding antibody wasused as a negative control (lack of competition).

FIG. 9. Internalization by macropinocytosis. YS5 IgG1 was incubated withmetastatic castration resistant prostate cancer cell line Du-145 alongwith the macropinocytosis indicator ND70-TRITC (Life Technologies) for 4h and 24 h respectively. Co-localization was analyzed by an Olympusfluorescence confocal microscopy.

FIG. 10. Immunohistochemistry study of anti-CD46 antibodies on FDAstandard panel of frozen tissues for therapeutic antibody evaluation.Shading indicates levels of positive staining with placentaltrophoblasts being the strongest. Signals in non-shaded ones are eitherweak or non-detectable.

FIG. 11. HPLC analysis of anti-CD46 antibody drug conjugates. YS5 IgG1was conjugated to monomethyl auristatin (MMAF) via the mc-vc-pab linkerand analyzed by HIC. The number above peaks indicate the number of drugmolecules. On average, about three drug molecules were conjugated to anIgG molecule.

FIG. 12. Kill curve for prostate cancer cell lines LNCaP-C4-2b.

FIG. 13. Kill curve of anti-CD46 ADC (YS5) on metastatic castrationresistant prostate cancer cell line Du-145.

FIG. 14. In vivo tumor killing by anti-CD46 (YS5) ADC using subcutaneousprostate cancer xenograft models. LNCaP-C4-2B cells were implantedsubcutaneously in SCID mice. Injection started at day-12 at 5 mg/kg ADCand four injections were administered (every 4-5 days). Mice in theanti-CD46 ADC group are being monitored for extended periodpost-treatment. N=6.

FIG. 15. CD46 is highly expressed on multiple myeloma cell lineRPMI8226. Anti-CD46 antibody binds to both prostate cancer (LNCaP) andmultiple myeloma cells while the anti-PSMA antibody J591 binds only toprostate cancer cells.

FIG. 16. Anti-CD46 ADC (YS5) kills RPMI8226 cells in vitro. Ctr ADC: anon-binding human IgG1 conjugated to MMAF.

FIG. 17. In vivo anti-CD46 (YS5) ADC activity. RPMI8226-Luc cells werei.v. injected to NSG mice to create disseminated tumor xenograft.CD46-MMAF: YS5 ADC; IgG-MMAF: control ADC. A total of 4 injections weregiven every 4 days. Treatment started on day 10. Three mice inbortezomib-treated group did not last to day 31.

FIG. 18. Kaplan-Meier analysis of survival data for mice carryingRPMI8226 xenograft post YS5 ADC treatment.

FIG. 19. Kill curve of anti-CD46 (YS5) ADC on colorectal cancer cellline HT29.

FIG. 20. Anti-CD46 ADC (YS5) on MM1S xenograft.

FIG. 21. Kaplan-Meier analysis of mice bearing orthometastatic MM1.Sxenografts post ADC treatment. 100% of mice treated with 4 mg/kganti-CD46 ADC survived til the end of the experiment (day-212).Injection started on day-10 post implant. Anti-CD46 was injected ateither a single dose of 4 mg/kg or 4 times at two dosing levels (0.8mg/kg and 4 mg/kg). The control ADC (Ctr ADC) was injected 4 times at 4mg/kg.

FIG. 22. Kill curve of anti-CD46 (YS12) ADC on colorectal cancer cellline HT29.

FIG. 23. Kill curve of anti-CD46 (SB1HGNY) ADC on colorectal cancer cellline HT29.

FIG. 24. Kill curve of anti-CD46 YS5 ADC on pancreatic cancer cell lineMiaCaPa2.

FIG. 25. Kill curve of anti-CD46 YS5 ADC on mesothelioma cell line M28.

FIG. 26. Kill curve of anti-CD46 YS5 ADC on ovarian cancer cell lineOVCAR3.

FIG. 27. Anti-CD46 ADC has no effect on BPH-1 cells. BPH-1 cells expressvery low levels of CD46 and is not affected by YS5 ADC.

FIG. 28. Anti-CD46 (YS5) ADC on HS27 cells. Hs27 cells were seeded at3,000 cells per well.

FIG. 29. Anti-CD46 ADC (YS5) show little toxicity on normal CD3+ Tcells. 10,000 CD3+ T cells were seeded in 96-well plates and incubatedwith varying concentrations of YS5 ADCs at 37° C. for 96 h. Cellviability was assessed by the CCK-8 (Cell Counting Kit-8) (Dojindo).

FIG. 30. Anti-CD46 ADC has no effect on normal CD14-depleted PBMCs.10,000 cells were seeded in 96 well plates, and incubated with varyingconcentrations of ADC at 37° C. for 98 h. Cell viability was determinedby the CCK8 counting kit.

FIG. 31. Anti-CD46 ADC toxicity evaluation in transgenic mice expressinghuman CD46. Following i.v. injection of 6 mg/kg anti-CD46 and controlADCs, mice were followed for 14 days, sacrificed and major organsharvested for histological examinations.

FIG. 32 shows that CD46 ADC is effective in intra femoral prostatecancer xenograft model. The mCRPC cell line LnCaP-C4-2B that carried afirefly luciferase reporter was injected into the femur of mice. CD46ADC (YS5-mcvcpab-MMAF) was injected on day 7 every 4 days for a total of4 injections. The mice were monitored post-treatment until day 65. CtrADC: a non-binding IgG1 conjugated to MMAF (ctr IgG-mcvcpab-MMAF).

FIG. 33 illustrates CD46 expression in castration resistant prostatecancer (CRPC). Prostate tissue specimens were taken from patients whobecame resistant to hormone blockage. Arrows indicate tumor cells (onlyselective tumor regions are indicated). The H-294 rabbit anti-human CD46antibody (Santa Cruz Biotechnology) was used for staining, followed bydetection with the Envision+ system (Dako North America).

FIG. 34 illustrates bone metastasis of mCRPC. Arrows indicate tumorcells (only selective tumor regions are indicated). The H-294 rabbitanti-human CD46 antibody (Santa Cruz Biotechnology) was used forstaining, followed by detection with the Envision+ system (Dako NorthAmerica).

FIG. 35 illustrates lymph node metastasis of mCRPC. Arrows indicatetumor cells (only selective tumor regions are indicated). The H-294rabbit anti-human CD46 antibody (Santa Cruz Biotechnology) was used forstaining, followed by detection with the Envision+ system (Dako NorthAmerica).

FIG. 36 illustrates bladder metastasis of mCRPC. Arrows indicate tumorcells (only selective tumor regions are indicated). The H-294 rabbitanti-human CD46 antibody (Santa Cruz Biotechnology) was used forstaining, followed by detection with the Envision+ system (Dako NorthAmerica).

FIG. 37 shows that CD46 is highly expressed by prostate cancerneuroendocrine cell line H660. Left panel: FACS analysis. Ctr:non-binding mAb. Right panel: Western blot analysis confirms CD46expression, and the expression of neuroendocrine marker NSE by H660cells.

FIG. 38 shows internalization of anti-CD46 antibody by prostate cancerneuroendocrine cell line H660. YS5 IgG1 was incubated with H660 cellsovernight. The cells were fixed, permeated, stained, and imaged byconfocal microscopy. A single confocal slice is shown. The anti-CD46antibody is internalized and co-localize with LAMP1, the lysosomemarker.

FIG. 39 shows that CD46 ADC kills H660 cells. Varying concentrations ofCD46 ADC (YS5-mcvcpab-MMAF) were incubated with the neuroendocrine cellline H660 at 37° C. for 7 days. Calcein AM assay was used to assessviability. Ctr ADC: a non-binding IgG1-mcvcpab-MMAF.

FIG. 40 shows that treatment of the metastatic castration resistantprostate cancer cell line LNCaP-C4-2B with 10 μM abiraterone (abi) for 7days caused upregulation of cell surface CD46 expression as measured byFACS. MFI: mean fluorescence intensity.

FIG. 41 shows that abiraterone (Abi)-treated LNCaP C4-2B cells are moresensitive to CD46 ADC. LNCaP-C4-2B cells were incubated with abirateronefor 7 days, washed and continued incubated with CD46 ADC in mediacontaining no abiraterone for an additional 96 hours. C4-2B CD46 ADC:LNCaP C4-2B cells incubated with CD46 ADC (YS5-mcvcpab-MMAF) withoutprior exposure to abiraterone (EC50=169 pM). C4-2B ABI CD46 ADC: LNCaPC4-2B cells with prior exposure to abiraterone incubated with CD46 ADC(EC50=21 pM). Ctr ADC: a non-binding IgG1 conjugated to MMAF.

FIG. 42 illustrates upregulation of cell surface CD46 on neuroendocrinecell line H660 post enzalutamide (ENZ) treatment. H660 cells weretreated with 10 μM enzalutamide for 7 days, and analyzed by FACS forcell surface antigen expression. CD46 is highly expressed by H660, whileprostate specific membrane antigen (PSMA) is barely detectable.Moreover, exposure to enzalutamide caused an upregulation of CD46expression on tumor cell surface. MFI: median fluorescence intensity.

FIG. 43 shows that CD46 is highly expressed in primary colorectalcancer. Arrows indicate tumor cells (only selective tumor regions areindicated). The H-294 rabbit anti-human CD46 antibody (Santa CruzBiotechnology) was used for staining, followed by detection with theEnvision+ system (Dako North America). Images were taken using a digitalmicroscope at two levels of amplification (4× and 20×).

FIG. 44 shows that CD46 is highly expressed in colorectal cancermetastasized into the liver. Arrows indicate tumor cells (only selectivetumor regions are indicated). The H-294 rabbit anti-human CD46 antibody(Santa Cruz Biotechnology) was used for staining, followed by detectionwith the Envision+ system (Dako North America). Images were taken usinga digital microscope at two levels of amplification (4× and 20×).

FIG. 45 shows that CD46 is highly expressed in colorectal cancermetastasized into the lymph node. Arrows indicate tumor cells (onlyselective tumor regions are indicated). The H-294 rabbit anti-human CD46antibody (Santa Cruz Biotechnology) was used for staining, followed bydetection with the Envision+ system (Dako North America). Images weretaken using a digital microscope at two levels of amplification (4× and20×).

FIG. 46 shows that CD46 is highly expressed in colorectal cancermetastasized to the bladder. Arrows indicate tumor cells (only selectivetumor regions are indicated). The H-294 rabbit anti-human CD46 antibody(Santa Cruz Biotechnology) was used for staining, followed by detectionwith the Envision+ system (Dako North America). Images were taken usinga digital microscope at two levels of amplification (4× and 20×).

FIG. 47 shows that CD46 is highly expressed in mesothelioma asdemonstrated by immunohistochemistry of CD46 staining in mesothelioma.Arrows indicate tumor cells (only selective tumor regions areindicated). The H-294 rabbit anti-human CD46 antibody (Santa CruzBiotechnology) was used for staining, followed by detection with theEnvision+ system (Dako North America).

FIG. 48 shows that CD46 is highly expressed in pancreatic cancer. Arrowsindicate tumor cells (only selective tumor regions are indicated). TheH-294 rabbit anti-human CD46 antibody (Santa Cruz Biotechnology) wasused for staining, followed by detection with the Envision+ system (DakoNorth America).

FIG. 49 shows that CD46 is overexpressed by glioblastoma multiforme(GBM) as illustrated by immunohistochemistry analysis of CD46 stainingin GBM and normal brain. No CD46 staining was observed in normal humanbrain but strong staining was observed in GBM specimens. The H-294rabbit anti-human CD46 antibody (Santa Cruz Biotechnology) was used forstaining, followed by detection with the Envision+ system (Dako NorthAmerica).

FIG. 50 illustrates in vivo inhibition of mesothelioma (M28) xenograftgrowth by CD46 ADC. YS5-mcvcpab-MMAF was injected every 3-4 days at 5mg/kg for a total of 5 times (indicated by arrows). Tumor volumes weremeasured by caliper. YSC10 is a control non-binding human IgG1.

DETAILED DESCRIPTION

In various embodiments, a number of new anti-CD46 antibodies areprovided. The prototypical antibodies described herein were identifiedby a combination of selections on cell surface and recombinant CD46using both phage and yeast display techniques. These antibodies areinternalized by the tumor-selective macropinocytosis pathway, withoutthe need of crosslinking and localize to the lysosomes, which makes themwell suited for the development of antibody drug conjugates (ADCs) andother targeted therapeutics that utilize intracellular payload release.

The antibodies described herein antibodies bind to domain 1 and 2 of theCD46 molecule, not the main complement binding domains 3 and 4, and thusdo not block directly the normal complement cascade.

Fine epitope mapping also showed differences in antigen contact sitesbetween the anti-CD46 antibodies described herein and the UA20 and 2B10antibodies described in PCT Application No: PCT/US2008/076704, andcopending U.S. application Ser. No. 14/205,101, respectively. Tested onCHO cells transfected with cynomolgus monkey CD46 cDNA, we found thatour anti-CD46 antibodies bind to an epitope conserved between human andcynomolgus monkey, thus identifying an appropriate species forregulatory toxicology study.

Using FDA-approved frozen human tissue panel for therapeutic antibodyevaluation, it was found that the CD46 epitopes bound by antibodies theantibodies described herein are expressed at low levels in virtually notissues except for placental trophoblasts and, to a lesser degree,prostate epithelium. Conversely, it was determined that CD46 isoverexpressed by a variety of tumors including but not limited to,prostate cancer, multiple myeloma, colorectal cancer, pancreatic cancer,mesothelioma, lung cancer, breast cancer, ovarian cancer, liver cancer,glioma, neuroblastoma, etc.

Given that CD46 is located in chromosome 1q32.2, and 1q gain has beenobserved in a broad spectrum of human cancers, it is likely to be anexcellent target for antibody therapy development for variousmalignancies. Antibody drug conjugates (ADCs) utilizing the anti-CD46antibodies described herein were constructed and it was found that theykill CD46-overexpressing cancer cell lines in vitro including but notlimited to metastatic castration resistant prostate cancer, multiplemyeloma, colorectal cancer, mesothelioma, ovarian cancer, etc. Mostimportantly, the anti-CD46 ADCs described herein showed potent in vivoanti-tumor activity, greatly reducing tumor burdens in xenograft modelsof castration resistant prostate cancer and multiple myeloma. Thispotent anti-tumor activity is believed to be applicable to otherCD46-overexpressing tumor models as well. The studies described hereinthus validate CD46 as a useful tumor cell surface antigen for targetedtherapy development. Additionally, the anti-CD46 antibodies describedherein can be used in companion diagnostics for patient stratificationand treatment outcome monitoring.

In view of these discoveries, it is believed that the anti-CD46antibodies described herein specifically bind and be internalized intocells that express or overexpress CD46. As CD46 isexpressed/overexpressed by a number of cancers including, but notlimited to ovarian cancer, breast cancer, lung cancer, prostate cancer,colon cancer, kidney cancer, pancreatic cancer mesothelioma, lymphoma,liver cancer, urothelial cancer, stomach cancer, and cervical cancer,these antibodies can be used to specifically target and internalize intothese and other CD46 positive cancer cells.

In certain embodiments these antibodies can be used without attachedeffectors for their intrinsic cytotoxic and/or cytostatic and/orantiproliferative activity on cells (particularly cancer cells). Incertain embodiments these antibodies can be attached to one or moreeffectors (e.g., second antibody, a detectable label, a cytotoxin, aliposome containing a drug, a radionuclide, a drug, a prodrug, a viralparticle, a cytokine, a chelate, etc.) to thereby form animmunoconjugate that will specifically bind and internalize into cancercells expressing or overexpressing CD46. In certain embodiments multipleeffectors will be attached to a single antibody, or in certainembodiments, multiple antibodies will be attached to a single effector,or in certain embodiments, a single antibody will be attached to asingle antibody.

In various embodiments methods of use of these antibodies and/orimmunoconjugates are provided. In certain embodiments the methodsinvolve contacting a cell that expresses or overexpresses CD46 (e.g., acancer cell such as an ovarian cancer cell, a breast cancer cell, a lungcancer cell, a prostate cancer cell, a colon cancer cell, a kidneycancer cell, a pancreatic cancer cell, etc.) with the constructresulting in internalization of the construct (or a portion thereof)into the cell and thereby delivering the effector to the target cell. Incertain embodiments the “contacting” comprises administering theantibody or the construct to a subject (e.g., a human or a non-humanmammal) in need thereof.

Antibodies that bind CD46

Antibodies were discovered that specifically bind CD46, in particulardomains 1 and/or 2, and that are internalized by prostate (and otherCD46 positive cancer cells) in situ, e.g., when the cancer cell is inthe tissue microenvironment. As indicated above, such antibodies areuseful for targeting cancers when used alone, or when attached to aneffector to form a “targeted effector”.

Accordingly in certain embodiments, an isolated antibody is providedthat that specifically binds CD46 and that is internalized into a cellthat expresses or overexpresses CD46 (e.g., a prostate cancer cell) viamacropinocytosis. In various embodiments, the antibody binds domain 1and/or domain 2 of CD46. In various embodiments, the antibody does notbind domain 3 and/or domain 4 of CD46.

The antibodies designated herein as YS5, YS5F, YS5v1D, SB1HGNY, YS12,3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY,3G7NY, 3G7, SB2, 2C8, and UA8kappa (see, e.g., Table 1) are illustrativeprototypical antibodies. In certain embodiments antibodies that compriseVL CDR1 and/or VL CDR2, and/or VL CDR3, and/or VH CDR1 and/or VH CDR2,and/or VH CDR3 of one or more of these antibodies are contemplated. Incertain embodiments antibodies that comprise the VH domain and/or the VLdomain of one or more of theses antibodies are contemplated. Alsocontemplated are antibodies that compete for binding at CD46 with one ormore of as YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1,YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/orUA8kappa.

The amino acid sequences of the VH and VL domains of YS5, YS5F, YS5v1D,SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10,YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa antibodies are shownin Table 1 (see Example 1).

TABLE 1 Novel human anti-CD46 antibody sequences. YS5 and YS5F differby one amino acid in VH CDR1 (L vs. F). YS5 and YS5vlD haveidentical VH but one amino acid difference in the VL CDR2(N vs. D). 3G7HY, 3G7NY, 3G7RY (aka 3G8), and 3G7 have one residue difference in VH CDR3, but entirely different VLs.YS6 and 3G7 have identical VH but different VL. VH VL YS5QVQLVQSGGGVVQPGRSLRLACAASGLTV QSVLTQPPSVSGAPGQRVTISCTGSSSNIGANNYAMHWVRQAPGKGLEWVAVISYDGNNK GYDVHWYQQLPGTAPKLLIYGNNNRPSGVPDYYADSVKGRFTISRDNSKNTLYLQMNSLR RFSGSKSGTSASLAITGLQAEDEADYYCSSYAEDTAVYYCAKGGGYFDLWGRGTLVTVSS TSGTWLFGGGTKLTVL (SEQ ID NO: 1)(SEQ ID NO: 22) YS5F QVQLVQSGGGVVQPGRSLRLACAASGFTVQSVLTQPPSVSGAPGQRVTISCTGSSSNIGA NNYAMHWVRQAPGKGLEWVAVISYDGNNKGYDVHWYQQLPGTAPKLLIYGNNNRPSGVPD YYADSVKGRFTISRDNSKNTLYLQMNSLRRFSGSKSGTSASLAITGLQAEDEADYYCSSY AEDTAVYYCAKGGGYFDLWGRGTLVTVSSTSGTWLFGGGTKLTVL (SEQ ID NO: 2) (SEQ ID NO: 23) YS5vIDQVQLVQSGGGVVQPGRSLRLACAASGFTV QSVLTQPPSVSGAPGQRVTISCTGSSSNIGANNYAMHWVRQAPGKGLEWVAVISYDGNNK GYDVHWYQQLPGTAPKLLIYGDNNRPSGVPDYYADSVKGRFTISRDNSKNTLYLQMNSLR RFSGSKSGTSASLAITGLQAEDEADYYCSSYAEDTAVYYCAKGGGYFDLWGRGTLVTVSS TSGTWLFGGGTKLTVL (SEQ ID NO: 3)(SEQ ID NO: 24) SB1HG QVQLQQSGGGVVQPGRSLRLSCAASGFTFDIQMTQSPSFLSASVGDRVTITCRASQGISS NY SSYAMHWVRQAPGKGLEWVAFIRSDGSKKYLAWYQQKPGKAPKLLIYAASTLQSGVPSSF YYADSVKGRFTISRDNSKNTLYLQMNSLRSGSGSGTEFTLTISSLQPEDFATYYCQQLAS AEDTAVYYCARHGNYFDSWGQGTLVTVSSYPLTFGGGTKVDIK (SEQ ID NO: 4) (SEQ ID NO: 25) YS12QVQLVESGGGVVQPGRSLRLSCAASGFTF SSELTQDPAVSVALGQTVRITCQGDSLRSYYSTYGMHWVRQAPGKGLEWLSFISYDGDEK VSWFQQKPGQAPVFVMYGQNNRPSGISERFSYYADSVKGRFTISRDNSKNTLYLQMNSLR GSSSGNTASLIITGAQAEDEADYYCHSRDSSAEDTAVYWCAKASGYGMGILDYWGQGTLV GTHLRVFGGGTKLTVL TVSS (SEQ ID NO: 26)(SEQ ID NO: 5) 3G7RY EVQLVESGGGLVQPGGSLRLSCAASGFTFQSALTQPPSASATPGQRVTISCSGRTSNIGS aka SDYYMSWIRQAPGKGLEWVSYISSSGSTINHVYWYQQLPGTAPKLLIYRNNQRPSGVPDR 3G8 YYADSVKGRFTISRDNSKNTLYLQMNSLRFSGSKSGTSASLAISGLRSEDEADYYCATWD AEDTAVYYCARDYGRIAAAGRRYWGQGTLDSLSGEVFGGGTKLTVL VTVSS (SEQ ID NO: 27) (SEQ ID NO: 6) YS6QVQLQESGGGVVRPGGSLRLSCAASGFTF SSELTQDPAVSVALGQTVRITCQGDSLRSYYSDYYMSWIRQAPGKGLEWVSYISSSGSTI ASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSYYADSVKGRFTISRDNSKNTLYLQMNSLR GSSSGNTASLTITGAQAEDEADYYCNSRDSSAEDTAVYYCARDYGRIAAAGRHYWGQGTL GTHLEVFGGGTKVTVL VTVSS (SEQ ID NO: 28)(SEQ ID NO: 7) YS1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSELTQDPAVSVALGQTVRITCQGDTLSTYY SDYYMSWIRQAPGKGLEWVSYISSSGSTIANWYQQKPGQAPVLVIYGKNNRPSGIPDRFS YYADSVKGRFTISRDNSKNTLYLQMNSLRGSSSGNTASLTITGAQAEDEADYYCHSRDIS AEDTAVYYCARDYGRIAAAGRHYWGQGTLGNYLFASGTKLTVL VTVSS (SEQ ID NO: 29) (SEQ ID NO: 8) YS3QVQLQESGGGLVQPGGSLRLSCAASGFTF QSVLTQPPSASGTPGQRVTISCSGSSSNIGSSSYWMSWVRQAPGKGLEWVADIKQDGSEK NTVNWSRQLPGTAPKLLIYSNNQRPSGVPDRYYVDSVKGRFTISGDNAKNSLYLQMNSLR FSGSKSGTSASLAISGLQSEDEADYYCAAWDAEDTAVYYCAKDVGSTAINYVRAYTWFDP DSLNVYVFGTGTKVTVL WGQGTLVTVSS(SEQ ID NO: 30) (SEQ ID NO: 9) YS4 QVQLQESGGGLVQPGGSLRLSCAASGFTFKIVLTQSPSSLSASVGDTVTIACRASRDIRN SNYAMSWVRQAPGKGLEWVSTISGSGSSTDLAWYQQKPGKAPKLLIYGASSLQSGVPSRF FYVDSVKGRFTISRDNSKNTLYLQMNSLRSGSGSGTEFILTISSLQPEDFATYYCHRLNS AEDTAVYYCAQGLYSSGWANWFDPRGQGTYPLTFGGGTKVDIK LVTVSS (SEQ ID NO: 31) (SEQ ID NO: 10) YS8QVQLQESGGGVVQPGRSLRLSCAASGFTF NFMLTQPASLSGSPGQSITISCTGTSSDVGGSSYGMHWVRQAPGKGLEWVAVISYDGSNK YNYVSWYQQHPGYAPKLMIYDVSNRPSGVSNYYADSVKGRFTISRDNSKNTLYLQMNSLR RFSGSKSGNTASLTISGLQAEDEADYYCSSYAEDTAVYYCAKVMGLAAAGLDAFDIWGQG TSSSTPWVFGGGTKLTVL TTVTVSS (SEQ ID NO: 32)(SEQ ID NO: 11) YS7 QVQLVQSGGGVVQPGRSLRLSCAASGFTFSYVLTQDPAVSVALGQTVRITCQGDSLRSYY SSYAMHWVRQAPGKGLEWVAVISYDGSNKASWYQQKPGQAPVLVIYGKNNRPSGIPDRFS YYADSVKGRFTISRDTSTNTLYLQMNSLRGSSSGNTASLTITGAQAEDEADYYCNSRDSS ADDTAVYYCGRESSGSPGVWGQGTTVTVSGNQFGGGTKLTVL S (SEQ ID NO: 33) (SEQ ID NO: 12) YS9QVQLVESGGGLIQPGGSLRLSCAASGFTV SSELTQDPAVSVALGQTVRITCQGDSLRTYYSSNYMSWVRQAPGKGLEWVSVIYTDGSTY ASWYQQRPGQAPILVLYGKNNRPSGIPDRFSYADSVKGRFTISRDNSKNTLYLQMNSLRA GSSSGNTASLTITGAQAEDEADYYCNSRDSSEDTAIYYCARDRGTSGYDWAWFDLWGQGT GNHVVFGGGTKLTVL LVTVSS (SEQ ID NO: 34)(SEQ ID NO: 13) YS10 QVQLQESGGGLVQPGGSLRLSCAASGFTFQSVLTQPASVSGSPGQSITISCTGTGSDVGS SSYAMSWVRQAPGKGLEWVSAISGSGGSTYNYVSWYQQNPGKAPKLMIYEVSNRPSGVSN YYADSVKGRFTISRDNSKNTLYMQMNSLRRFSGSKSGNTASLTISGLQAEDEADYYCSSY AEDTAVYYCAKDRYYYGSGKDAFDIWGRGTTSSTLVFGGGTKVTVL TMVTVSS (SEQ ID NO: 35) (SEQ ID NO: 14) YS11QVQLVESGGGLVQPGGSLGLSCAASGFTF SELTQDPAVSVALGQTVRITCQGDSLRSYYASNYWMSWVRQAPGKGLEWVANVRQDGGQK SWYQQKPGQAPVLVIYGENSRPSGIPDRFSGYYVDSVKGRFTISRDNAKNSLYLQMNSLR SSSGNTASLTITGAQAEDEADYYCNSWDSSGTEDTAVYFCVSQRNSGEHDYWGQGTLVTV NHVVFGGGTKLTVL SS (SEQ ID NO: 36)(SEQ ID NO: 15) 3G7HY EVQLVESGGGLVQPGGSLRLSCAASGFTFAIRMTQSPSSLSASVGDRVTITCRASQSISS SDYYMSWIRQAPGKGLEWVSYISSSGSTIYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF YYADSVKGRFTISRDNSKNTLYLQMNSLRSGSGSGTDFTLTISSLQPEDFATYYCQQSYS AEDTAVYYCARDYGRIAAAGRHYWGQGTLTPRTFGQGTKLEIK VTVSS (SEQ ID NO: 37) (SEQ ID NO: 16) 3G7NYEVQLVESGGGLVQPGGSLRLSCAASGFTF DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDYYMSWIRQAPGKGLEWVSYISSSGSTI SNGYDYLDWYLQKPGQSPQLLIYLGSNRASGYYADSVKGRFTISRDNSKNTLYLQMNSLR VPDRFSGSGSGTDFTLKISRVETEDVGIYYCAEDTAVYYCARDYGRIAAAGRNYWGQGTL MQGLQTPSFGQGTKLEIK VTVSS (SEQ ID NO: 38)(SEQ ID NO: 17) 3G7 QVQLQESGGGVVRPGGSLRLSCAASGFTFSSELTQDPAVSVALGQTVRITCQGDSLRSYY SDYYMSWIRQAPGKGLEWVSYISSSGSTIASWYQQKPGQAPVPVIYGKNNRPSGIPDRFS YYADSVKGRFTISRDNSKNTLYLQMNSLRGSSSGNTASLTITGAQAEDEADYYCNSRDSS AEDTAVYYCARDYGRIAAAGRHYWGQGTLSTHRGVFGGGTKLTVL VTVSS (SEQ ID NO: 39) (SEQ ID NO: 18) SB2EVQLVESGGGLVKPGGSLRLSCAASGFTF DIQLTQSPSSLSASVGDRVTITCRASRSISTSDYYMSWIRQAPGKGLEWVSYISSSGSSI YLSWYQQKPGKAPKLLIYDASRLQNGVPSRFYYADSVKGRFTISRDNAKNSLYLQMNSLK SGSGSDTDFTLTISSLQPEDFATYFCQQSYNAEDTAVYYCARDITDVVGVSFDYWGQGTL PPWTFGQGTKLEIK VTVSS (SEQ ID NO: 40)(SEQ ID NO: 19) 2C8 EVQLVESGGGVVQPGRSLRLSCAASGFTFQSALTQPASVSGSPGQSITISCTGTSSDVGG SSYGMHWVRQAPGKGLEWVAVISYDGSNKYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSN YYADSVKGRFTISRDNSKNTLYLQMNSLRRFSGSKSGNTASLTISGLQAEDEAYYYCSSY AEDTAEYYCAKVMGLAAAGLDAFDIWGQGTSSSDPWVFGGGTQLTVL TLVTVSS (SEQ ID NO: 41) (SEQ ID NO: 20) UASkapEVQLVESGGGVVQPGRSLRLSCAASGFTF NIQMTQSPSSLSASVGDRVTITCRAGQPIST paSSFGMHWVRRAPGKGLEWVAVISYDGSNQ YVNWYQHKPGKAPKLLIYGASNLQSGVPSRFYYADSVKGRFTISRDNSKNTLYLQMNSLR SGGGSATDFTLTISSLQPEDFATYYCQQSYSAEDTAVYYCGSRPGGGYASGSTVAYWGQG SLLTFGDGTKVEIK TLVTVSS (SEQ ID NO: 42)(SEQ ID NO: 21)

In various embodiments the antibodies contemplated herein expresslyexclude antibodies composing the three VH CDRs and/or the three VL CDRsof antibodies 3051.1, G12FC3, M6c42b, 4F3YW, M40pr146, UA20, UA8,585II41, 585II41.1, 585II56, 3076, 3051, M49R, RCI-14, II79_4, II79_3,T5II-4B.1, T5II-4B.2, RCI-11, RCI-20, CI-11A, CI-14A, S95-2 that aredescribed in PCT/US2008/076704 (WO 2009/039192) and/or the mPA7antibody. The amino acid sequences of the VH and VL chains of theseantibodies and the CDRs comprising these domains are shown in inPCT/US2008/076704 and the amino acid sequences of these domains arereproduced below in Table 2.

TABLE 2 Excluded antibodies. The sequence shown below are scFvantibodies (the VL and VH regions are joined by a(Gly₄Ser)₃ (SEQ ID NO: 43) linker, however it willbe recognized that other antibody forms comprising the CDRs(or the VH and/or VL domains) are similarly excluded. CloneAmino Acid Sequence SEQ ID No 3051.1QVQLQESGGGLVKPGGPLRLSCAASGFTFSSYGMYWVRQAPGKGLEWV 4STLSRSGSGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ASIAVAGNYFDYWGQGTLVTVSSGGGGSGGGGSGGGGS SYVLTQDPAVSVALGQTVRITCQGDSLRSYYASWYQERPGQAPLLVIYGKNNRPSGIPDRFSGSNSGSTATLTISRVEAGDEGDYYCQVWDSINEQVVFGGGTKVT VL G12FC3QVQLVQSGGGVVQPGRSLRLSCAATGIPFSGSGMHWVRQAPGKGLEWV 5TMIWYDGSNKFYADSVKGRFTISRDNSKNTLYLQMDSLRAEDTAVYFC ARDKGVRSMDVWGLGTTVTVSSGGGGSGGGGSGGGGS NFMLTQPPSVSVAPGQTAKITCDGYSIRTKSVHWYQQKPGQAPVVVVHDDSDRPSGIPERFSGSNSGTTATLTISRVEAGDEADYYCQAWDSISEEVVFGGGTKLTV L M6c42bQVQLQESGGGLVQPGGSLRLSCSASGFTFGTYAMRWVRQTSGKGLEWV 6SGIGVSGDAYYTDSVRGRFTISRDNSKNTLYLQMNTLRAEDTATYYCT RKSSTTSNDYWGRGTLVTVSS GGGGSGGGGSGGGGS SYVLTQDPAVSVALGQTVRITCQGDNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGTTATLTISSVEAGDEADYYCQAWDSISEHVIFGGGTKVTVL 4F3YWQVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWV 7AVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARFSSGWYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGS DIQMTQSPSFLSASVGDRITITCRASHDISSYFAWYQQKPGKAPKPLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLGSYPLTFGGGTKLEI K M40pr146QVQLLQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV 8SAISGSGGSTYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSHDYGDYAGFDYWGQGTLVTVSS GGGGSGGGGSGGGGS HVILTQDPAVSVALGQTVRITCQGDSLKSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGTTASLTITGAQAEDEADYYCHSRDSSGTHLRVFGGGT KLTVL UA20QVQLQESGGGLVKPGGSLRLSCAASGFTFSNAWMNWVRQAPGKGLEWV 9GRIKSKTDEGTTDYAAPVKGRFSISRDDSKNTLYLQMNSLKTEDTGVY YCTATKGLGGSKLGQGTLVTVSSGGGGSGGGGSGGGGS QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNTVNWSRQLPGTAPKLLIYSNDQRPSGVPDRFSGSKSGTSASLAITGLQPEDEADYYCGTWDSSLSAYVFGTGTK LTVL UA8QVQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMHWVRRAPGKGLEWV 0AVISYDGSNQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGSRPGGGYASGSTVAYWGQGTPVTVSS GGGGSGGGGSGGGGS SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPLLVIYGQNIRPSGIPDRFSGSSSGNSASLTITGAQAEDEADYYCHSRDSSGKYVFGVGT KVTVL 585II41QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMGWVRQAPGKGLEWV 1SAISGSGGSTYYADSVKGRFTISRDNSKDTLYLQMNSLRAEDTAVYYC ASRSLLDYWGQGTLVTVSSGGGGSGGGGSGGGGS NFMLTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPLLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNPVFGGGTKVTVL 585II41.1QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV 2SAISGSGGSTYYADSVKGRFTISRDNSKDTLYLQMNSLRAEDTAVYYC ASRSLLDYWGQGTLVTVSSGGGGSGGGGSGGGGS NFMLTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPLLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNPVFGGGTKVTVL 585II56QVQLQESGGGLVQLGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV 3SAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAFYYC ANSAYTGGWYDYWGHGTLVTVSSGGGGSGGGGSGGGGS SSELTQDPAVSVALGQTVKITCQGDSLRTYYASWYQQRPGQAPVLVIYGENSRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHLRVFGGGTKL TVL 3076QVNLRESGGGLVQPGGFLRLSCAAFGFTFSGYWMSWVHPAPGKGLEWV 4ANIKQDGSEKFYVDSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYFC ARGLLSDYWGQGTLVPVSSGGGGSGGGGSGGGGS NFMLTQPPSVSVAPGKTASLTCGGYNIGTKSVHWYQQKPGQAPVVVVHDDSDRPSGIPERFSGSNSGTTATLTIIRVEAGDEADYYCQAWDSISEEVVFGGGTKLTVL 3051QVQLQESGGGLVKPGGPLRLSCAASGFTFSSYGMYWVRQAPGKGLEWV 5STLSRSGSGTYYAESVKGRFTISRDNSKNTLYFQMNSLRAEDTAVYYC ASIAVAGNYFEYWGQGTLVTVSSGGGGSGGGGSGGGGS SYVLTQDPAVSVALGQTVRITCQGDSLRSYYASWYQERPGQAPLLVIYGKNNRPSGIPDRFSGSNSGSTATLTISRVEAGDEGDYYCQVWDSINEQVVFGGGTKVT VL M49RQVQLQESGGGLVKPGESLRLSCAASGFTFSDHYMDWVRQAPGKGLEWV 6AYIRYDGSTKYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAFYYCARLIAEAEGWFDPWGQGTLVTVSS GGGGSGGGGSGGGGS NFMLTQPPSVSVAPGKTARITCGGNNIGSKSVYWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKV TVL RCI-14QVQLLQSAGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWV 7SGISGSGGSTNYADSVKGRFTISRDSSKNTLFLQMNSLRAEDTAVYYC AKDYGSGWYDYWGQGTLVTVSSGGGGSGGGGSGGGGS SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQERPGQAPLLVIYGRNERPSGIPDRFSASSSGNTASLTITGAQAEDEADYYCQVWDSFNEQVVFGGGTKLTV L II79_4QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVHQAPGKGLEWV 8SAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTYYGFWSGYYDYLGQGTLVTVSS GGGGSGGGGSGGGGS SSELTQDPAVSVGLGQTVTITCQGDSLRSYYANWYQQKPGQAPILVIYGENNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCHSRDSSGTHLRVFGGGT KLTVL II79_3QVQLLESGGGVVQPGTSLRLSCAASGFTFSNYAINWVRQAAGKGLEWV 9SGISGSGVSTSYADSVKGRFTVSRDNSKNTLYLQMNSLRVEDTALYYC AKNGGGPEYLQHWGQGTLVTVSSGGGGSGGGGSGGGGS QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNTVNWSRQLPGTAPKLLIYSNDQRPSGVPDRFSGSKSGTSASLAITGLQPEDEADYYCGTWDSSLSAYVFGTGTK LTVL T5II-4B.1QVQLQESGGTLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGRGLEWV 0STISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKGAYSGSYWGQGTLVTVSSGGGGSGGGGSGGGGS SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPSLVIYGENSRPSGIPDRFSGSSSGNTASLTITGAQAENEADYYCQAWDSSTAVVFGGGTKLTVL T5II-4B.2QVQLQESGGTLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGRGLEWV 1STISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKGAYSGSHWGQGTLVTVSSGGGGSGGGGSGGGGS SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPSLVIYGENSRPSGIPDRFSGSSSGNTASLTITGAQAENEADYYCQAWDSSTAVVFGGGTKLTVL RCI-IIQVQLVESGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWM 2GWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARPIYDSSGYDAFDIWGQGTMVTVSS GGGGSGGGGSGGGGS DIVMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQYHTISRTFGPGTK VDIK RCI-20QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWV 3AVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFC VRPSDSGWSFEHWGQGTLVPVSSGGGGSGGGGSGGGGS QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNTVNWSRQLPGTAPKLLIYSNDQRPSGVPDRFSGSKSGTSASLAITGLQPEDEADYYCGTWDSSLSAYVFGTGTK LTVL CI-IIAQVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV 4AVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRGDRSYGAEYFQHWGQGTLVTVSSGGGGSGGGGSGGGGS SSELTQDPAVSVASGQTVRITCQGDSLRSYYASWYQQKPGQAPLLVIYGKNIRPSGIPDRFSGSTSGNSASLTITGAQAEDEADYYCNSRDSSGNRNWVFGGGT KLTVL CI-14AQVQLQESGGGLVKPGGSLRLSCAASGFTSSSYAMHWVRQAPGKGLEYV 5SAIGGNGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEGEQWLEYRYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPSLVIYGENSRPSGIPDRFSGSSSGNTASLTITGAQAENEADYYCQAWDSSTAVVFGGG TKLTVL S95-2QVQLVESGGGVVQPGRSLRLSCTASGFTFSSYGMHWVRQAPGKGLEWV 6AVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGRYSSNWFSYYYYGMDVWGQGTTVTVSS GGGGSGGGGSGGGGS NFMLTQPPSVSVAPGKTARITCGGNNIGSKSVYWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVV FGGGTKVTVL

Using the amino acid sequences provided for the YS5, YS5F, YS5v1D,SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10,YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and UA8kappa antibodies, numerousantibody forms can be prepared, e.g., as described below. Such formsinclude, but are not limited to a substantially intact (e.g., fulllength) immunoglobulin (e.g., an IgA, IgE, IgG, and the like), anantibody fragment (e.g., Fv, Fab, (Fab′)₂, (Fab′)₃, IgGACH₂, a minibody,and the like), a single chain antibody (e.g., scFv), a diabody, aunibody, an affibody, and the like.

It will be recognized, that where the antibodies are single chainantibodies, the VH and VL domains comprising such antibody can be joineddirectly together or by a peptide linker. Illustrative peptide linkersinclude, but are not limited to GGGGS GGGGS GGGGS (SEQ ID NO:67), GGGGSGGGGS (SEQ ID NO:68), GGGGS (SEQ ID NO:69), GS GGGGS GGGGS GGS GGGGS(SEQ ID NO:70), SGGGGS (SEQ ID NO:71), GGGS (SEQ ID NO:72), VPGV (SEQ IDNO:73), VPGVG (SEQ ID NO:74), GVPGVG (SEQ ID NO:75), GVG VP GVG (SEQ IDNO:76), VP GVG VP GVG (SEQ ID NO:77), GGSSRSS (SEQ ID NO:78), andGGSSRSSSSGGGGSGGGG (SEQ ID NO:79), and the like.

As indicated above, in various embodiments, the antibody binds (e.g.,specifically binds CD46 (e.g., domains 1 and/or 2). Typically antibodiescontemplated herein will specifically bind prostate cancer cellsincluding, but not limited to cells of a cell line selected from thegroup consisting of DU145 cells, PC3 cells, and LnCaP cells. In certainembodiments the antibody binds to a prostate tumor cell with an affinitygreater than (KD less than) about 5 nM when measured on live prostatetumor cells by FACS. In certain embodiments the affinity is greater than(K_(D) less than) about 1 nM, or at about 100 pM, or about 50 pM, orabout 10 pM, or about 1 pM.

Using the sequence information provided herein antibodies comprising oneor more of the CDRs comprising, e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12,3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY,3G7NY, 3G7, SB2, 2C8, and UA8kappa, or antibodies comprising the VHand/or VL domain(s) of these antibodies can readily be prepared usingstandard methods (e.g. chemical synthesis methods and/or recombinantexpression methods) well known to those of skill in the art, e.g., asdescribed below.

In addition, other “related” prostate cancer specific antibodies can beidentified by screening for antibodies that bind to the same epitope(e.g. that compete with one or more of YS5, YS5F, YS5v1D, SB1HGNY, YS12,3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY,3G7NY, 3G7, SB2, 2C8, and/or UA8kappa antibodies for binding to CD446and/or to a cell expressing or overexpressing CD46, e.g., a prostatecancer cell) and/or by modification of the YS5, YS5F, YS5v1D, SB1HGNY,YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11,3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa antibodies identifiedherein to produce libraries of modified antibody and then rescreeningantibodies in the library for improved binding to and/or internalizationinto cells expressing or overexpressing CD46, e.g., prostate cancercells.

Identification of Other Antibodies Binding the Same CD46 epitope(s) asYS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4,YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa.

Having identified CD46, especially domains one and/or two as a usefulantibody target and YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8),YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2,2C8, and UA8kappa antibodies as useful prototypical antibodies, other“related” antibodies that bind CD46 and preferably that are internalizedvia macropinocytosis can readily be identified by screening forantibodies that bind CD46 domains 1 and/or 2, e.g., by raising (e.g.,monoclonal antibodies) that specifically bind CD46 domains 1 and/or 2.Additionally or alternatively, other antibodies that bind CD46 and thatare internalized by macropinocytosis, can be identified by screening forantibodies that that cross-react with one or more of antibodies YS5,YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8,YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa,e.g., at the epitope bound by YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY(aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY,3G7, SB2, 2C8, and/or UA8kappa, and/or for antibodies that cross-reactwith one or more of YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8),YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2,2C8, and/or UA8kappa for binding to a prostate cancer cell (e.g., CaPcells, PC3 cells, etc.), and/or with an idiotypic antibody raisedagainst YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1,YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/orUA8kappa antibody.

Monoclonal Antibodies.

Monoclonal antibodies that bind CD46 domains 1 and/or 2, preferablybinding the epitope bound by one or more of YS5, YS5F, YS5v1D, SB1HGNY,YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11,3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa can be produced using avariety of known techniques, such as the standard somatic cellhybridization technique described by Kohler and Milstein (1975) Nature256: 495, viral or oncogenic transformation of B lymphocytes or phagedisplay technique using libraries of human antibody genes. In particularembodiments, the antibodies are fully human monoclonal antibodies.

Accordingly, in one embodiment, a hybridoma method is used for producingan antibody that binds CD46, preferably binding the domain 1 and/ordomain 2 of CD46. In this method, a mouse or other appropriate hostanimal can be immunized with a suitable antigen in order to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the antigen used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes canthen be fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding (1986) MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press)).Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen. Afterhybridoma cells are identified that produce antibodies of the desiredspecificity, affinity, and/or activity, the clones may be subcloned bylimiting dilution procedures and grown by standard methods (Id.).Suitable culture media for this purpose include, for example, D-MEM orRPMI-1640 medium. In addition, the hybridoma cells may be grown in vivoas ascites tumors in an animal. The monoclonal antibodies secreted bythe subclones can be separated from the culture medium, ascites fluid,or serum by conventional immunoglobulin purification procedures such as,for example, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

In another embodiment, antibodies and antibody portions that bind CD46domains 1 and/or 2 can be isolated from antibody phage librariesgenerated using the techniques described in, for example, McCafferty etal. (1990) Nature, 348: 552-554, Clackson et al. (1991) Nature,352:624-628, Marks et al. (1991) J. Mol. Biol., 222: 581-597, Hoet et al(2005) Nature Biotechnol., 23: 344-348; U.S. Pat. Nos. 5,223,409;5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 toMcCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731;6,555,313; 6,582,915 and 6,593,081 to Griffiths et al. Additionally,production of high affinity (nM range) human antibodies by chainshuffling (Marks et al. (1992) Bio/Technology, 10:779-783), as well ascombinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al. (1993) Nucl.Acids. Res., 21: 2265-2266) may also be used.

In a particular embodiment, the monoclonal antibody or antigen bindingportion thereof that binds CD46, preferably binding the epitope of boundby one or more of YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8),YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2,2C8, and/or UA8kappa is produced using the phage display techniquedescribed by Hoet et al., supra. This technique involves the generationof a human Fab library having a unique combination of immunoglobulinsequences isolated from human donors and having synthetic diversity inthe heavy-chain CDRs is generated. The library is then screened for Fabsthat bind to CD46, preferably competing for binding with one or more ofYS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4,YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa.

In yet another embodiment, human monoclonal antibodies directed againstCD46, preferably comprising the epitope bound by one or more of YS5,YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8,YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa canbe generated using transgenic or transchromosomic mice carrying parts ofthe human immune system rather than the mouse system (see e.g., Lonberg,et al. (1994) Nature 368(6474): 856-859; Lonberg and Huszar, (1995)Intern. Rev. Immunol. 13: 65-93, Harding and Lonberg (1995) Ann. NYAcad. Sci. 764: 536-546, and U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318;5,874,299; and 5,770,429; all to Lonberg and Kay; U.S. Pat. No.5,545,807 to Surani et al.; PCT Publication Nos. WO 92/03918, WO93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all toLonberg and Kay; and PCT Publication No. WO 01/14424 to Korman et al.).

In another embodiment, human antibodies directed against CD46 preferablybinding the epitope bound by one or more of YS5, YS5F, YS5v1D, SB1HGNY,YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11,3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa can be raised using a mousethat carries human immunoglobulin sequences on transgenes andtranschomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome (see e.g., PCTPublication WO 02/43478 to Ishida et al.).

Alternative transgenic animal systems expressing human immunoglobulingenes are available in the art and can be used to raise anti-CD46antibodies of the invention. For example, an alternative transgenicsystem referred to as the Xenomouse (Abgenix, Inc.) can be used; suchmice are described in, for example, U.S. Pat. Nos. 5,939,598; 6,075,181;6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.

Alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-CD46 antibodies contemplated herein. For example, mice carryingboth a human heavy chain transchromosome and a human light chaintranchromosome can be used; as described in Tomizuka et al. (2000) Proc.Natl. Acad. Sci. USA 97: 722-727. Furthermore, cows carrying human heavyand light chain transchromosomes have been described in the art (see,e.g., Kuroiwa et al. (2002) Nature Biotechnology 20: 889-894) and can beused to raise anti-CD46 CCP1 antibodies.

In yet another embodiment, antibodies that specifically bind CD46,preferably binding the epitope bound by one or more of YS5, YS5F,YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7,YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa can beprepared using a transgenic plant and/or cultured plant cells (such as,for example, tobacco, maize and duckweed) that produce such antibodies.For example, transgenic tobacco leaves expressing antibodies or antigenbinding portions thereof can be used to produce such antibodies by, forexample, using an inducible promoter (see, e.g., Cramer et al. (1999)Curr. Top. Microbol. Immunol. 240: 95-118). Also, transgenic maize canbe used to express such antibodies and antigen binding portions thereof(see, e.g., Hood et al. (1999) Adv. Exp. Med. Biol. 464: 127-147).

Antibodies can also be produced in large amounts from transgenic plantseeds including antibody portions, such as single chain antibodies(scFv's), for example, using tobacco seeds and potato tubers (see, e.g.,Conrad et al. (1998) Plant Mol. Biol. 38: 101-109). Methods of producingantibodies or antigen binding portions in plants can also be found in,e.g., Fischer et al. (1999) Biotechnol. Appl. Biochem. 30: 99-108, Ma etal. (1995) Trends Biotechnol. 13: 522-527, Ma et al. (1995) PlantPhysiol. 109: 341-346; Whitelam et al. (1994) Biochem. Soc. Trans. 22:940-944, and U.S. Pat. Nos. 6,040,498 and 6,815,184.

The binding specificity of monoclonal antibodies or portions thereofthat bind CD46, preferably comprising the preferably binding the epitopebound by one or more of YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7,SB2, 2C8, and/or UA8kappa prepared using any technique including thosedisclosed here, can be determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). The binding affinity of a monoclonalantibody or portion thereof also can be determined by the Scatchardanalysis of Munson et al. (1980) Anal. Biochem., 107:220.

Cross-Reactivity with Anti-idiotypic Antibodies.

The idiotype represents the highly variable antigen-binding site of anantibody and is itself immunogenic. During the generation of anantibody-mediated immune response, an individual will develop antibodiesto the antigen as well as anti-idiotype antibodies, whose immunogenicbinding site (idiotype) mimics the antigen.

Anti-idiotypic antibodies can be raised against the variable regions ofthe antibodies identified herein (e.g., YS5, YS5F, YS5v1D, SB1HGNY,YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11,3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa) using standard methodswell known to those of skill in the art. Briefly, anti-idiotypeantibodies can be made by injecting the antibodies of this invention, orfragments thereof (e.g., CDRs) into an animal thereby eliciting antiseraagainst various antigenic determinants on the antibody, includingdeterminants in the idiotypic region.

Methods for the production of anti-analyte antibodies are well known inthe art. Large molecular weight antigens (greater than approx. 5000Daltons) can be injected directly into animals, whereas small molecularweight compounds (less than approx. 5000 Daltons) are preferably coupledto a high molecular weight immunogenic carrier, usually a protein, torender them immunogenic. The antibodies produced in response toimmunization can be utilized as serum, ascites fluid, an immunoglobulin(Ig) fraction, an IgG fraction, or as affinity-purified monospecificmaterial.

Polyclonal anti-idiotype antibodies can be prepared by immunizing ananimal with the antibodies of this invention prepared as describedabove. In general, it is desirable to immunize an animal which isspecies and allotype-matched with the animal from which the antibody(e.g. phage-display library) was derived. This minimizes the productionof antibodies directed against non-idiotypic determinants. The antiserumso obtained is then usually absorbed extensively against normal serumfrom the same species from which the phage-display library was derived,thereby eliminating antibodies directed against non-idiotypicdeterminants. Absorption can be accomplished by passing antiserum over agel formed by crosslinking normal (nonimmune) serum proteins withglutaraldehyde. Antibodies with anti-idiotypic specificity will passdirectly through the gel, while those having specificity fornon-idiotypic determinants will bind to the gel. Immobilizing nonimmuneserum proteins on an insoluble polysaccharide support (e.g., sepharose)also provides a suitable matrix for absorption.

Monoclonal anti-idiotype antibodies can be produced using the method ofKohler et al. (1975) Nature 256: 495. In particular, monoclonalanti-idiotype antibodies can be prepared using hybridoma technologywhich comprises fusing (1) spleen cells from a mouse immunized with theantigen or hapten-carrier conjugate of interest (i.e., the antibodies orthis invention or subsequences thereof) to (2) a mouse myeloma cell linewhich has been selected for resistance to a drug (e.g., 8-azaguanine).In general, it is desirable to use a myeloma cell line which does notsecrete an immunoglobulin. Several such lines are known in the art. Onegenerally preferred cell line is P3X63Ag8.653. This cell line is ondeposit at the American Type Culture Collection as CRL-1580.

Fusion can be carried out in the presence of polyethylene glycolaccording to established methods (see, e.g., Monoclonal Antibodies, R.Kennett, J. McKearn & K. Bechtol, eds. N.Y., Plenum Press, 1980, andCurrent Topics in Microbiology & Immunology, Vol. 81, F. Melchers, M.Potter & N. L. Warner, eds., N.Y., Springer-Verlag, 1978). The resultantmixture of fused and unfused cells is plated out inhypoxanthine-aminopterin-thymidine (HAT) selective medium. Under theseconditions, only hybrid cells will grow.

When sufficient cell growth has occurred, (typically 10-14 dayspost-fusion), the culture medium is harvested and screened for thepresence of monoclonal idiotypic, anti-analyte antibody by any one of anumber of methods which include solid phase RIA and enzyme-linkedimmunosorbent assay. Cells from culture wells containing antibody of thedesired specificity are then expanded and recloned. Cells from thosecultures that remain positive for the antibody of interest are thenusually passed as ascites tumors in susceptible, histocompatible,pristane-primed mice.

Ascites fluid is harvested by tapping the peritoneal cavity, retestedfor antibody, and purified as described above. If a nonsecreting myelomaline is used in the fusion, affinity purification of the monoclonalantibody is not usually necessary since the antibody is alreadyhomogeneous with respect to its antigen-binding characteristics. Allthat is necessary is to isolate it from contaminating proteins inascites, i.e., to produce an immunoglobulin fraction.

Alternatively, the hybrid cell lines of interest can be grown inserum-free tissue culture and the antibody harvested from the culturemedium. In general, this is a less desirable method of obtaining largequantities of antibody because the yield is low. It is also possible topass the cells intravenously in mice and to harvest the antibody fromserum. This method is generally not preferred because of the smallquantity of serum which can be obtained per bleed and because of theneed for extensive purification from other serum components. However,some hybridomas will not grow as ascites tumors and therefore one ofthese alternative methods of obtaining antibody must be used.

Cross-Reactivity with the YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7,SB2, 2C8, and/or UA8kappa.

In another approach, antibodies that bind CD46 can be identified by thefact that they bind the same epitope as the “prototypic” antibodies ofthis invention (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8),YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2,2C8, and/or UA8kappa). To identify such antibodies, it s not necessaryto isolate the subject epitope. In certain embodiments, one can screen,e.g. antibody libraries for antibodies that compete with the prototypicantibodies of this invention for binding and/or internalization by aprostate cancer cell (e.g. a CaP cell, a PC3 cell, etc.), and/or forbinding to CD46.

Methods of screening libraries for epitope binding and/or cell bindingand/or internalization are well known to those of skill in the art. Incertain embodiments, cross-reactive prostate antibodies show at least60%, preferably 80%, more preferably 90%, and most preferably at least95% or at least 99% cross-reactivity with the one or more of the YS5,YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8,YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappaantibodies described herein.

Phage Display Methods to Select Other “Related” Anti-CD46 Antibodies.

Using the known sequences for the YS5, YS5F, YS5v1D, SB1HGNY, YS12,3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY,3G7NY, 3G7, SB2, 2C8, and/or UA8kappa and/or other prostate specificantibodies, a variety of phage display (or yeast display) methods can beused to generate other antibodies that antibodies that specifically bindCD46, preferably binding the epitope bound by YS5, YS5F, YS5v1D,SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10,YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa, with the same oreven greater affinity.

Chain Shuffling Methods.

One approach to creating antibody variants has been to replace theoriginal V_(H) or V_(L) gene with a repertoire of V-genes to create newpartners (chain shuffling) (Clackson et al. (1991) Nature. 352: 624-628)in a phage display or yeast display library. Using chain shuffling andphage display, the affinity of a human scFv antibody fragment that boundthe hapten phenyloxazolone (phOx) was increased from 300 nM to 1 nM (300fold) (Marks et al. (1992) Bio/Technology 10: 779-783).

Thus, for example, to alter the affinity of an anti-CD46 antibodydescribed herein, a mutant scFv gene repertoire can be createdcontaining a V_(H) gene of the prototypic YS5, YS5F, YS5v1D, SB1HGNY,YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11,3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa antibody (e.g. as shown inFIG. 2) and a human V_(L) gene repertoire (light chain shuffling). ThescFv gene repertoire can be cloned into a phage display vector, e.g.,pHEN-1 (Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133-4137) orother vectors, and after transformation a library of transformants isobtained.

Similarly, for heavy chain shuffling, a mutant scFv gene repertoire canbe created containing a V_(L) gene of the prototypic YS5, YS5F, YS5v1D,SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10,YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa antibody (e.g. asshown in FIG. 2) and a human V_(H) gene repertoire (heavy chainshuffling). The scFv gene repertoire can be cloned into a phage displayvector, e.g., pHEN-1 (Hoogenboom et al. (1991) Nucleic Acids Res., 19:4133-4137) or other vectors, and after transformation a library oftransformants is obtained.

The resulting libraries can be screened against the relevant target(e.g., CD46) and/or for cross-reactivity with YS5, YS5F, YS5v1D,SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10,YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa.

Site-Directed Mutagenesis to Improve Binding Affinity.

The majority of antigen contacting amino acid side chains are typicallylocated in the complementarity determining regions (CDRs), three in theV_(H) (CDR1, CDR2, and CDR3) and three in the V_(L) (CDR1, CDR2, andCDR3) (Chothia et al. (1987) J. Mol. Biol., 196: 901-917; Chothia et al.(1986) Science, 233: 755-8; Nhan et al. (1991) J. Mol. Biol., 217:133-151). These residues contribute the majority of binding energeticsresponsible for antibody affinity for antigen. In other molecules,mutating amino acids which contact ligand has been shown to be aneffective means of increasing the affinity of one protein molecule forits binding partner (Lowman et al. (1993) J. Mol. Biol., 234: 564-578;Wells (1990) Biochemistry, 29: 8509-8516). Site-directed mutagenesis ofCDRs and screening against the prostate cancer cells, in particular forbinding at CD46 e.g. as described herein in the examples, can produceantibodies having improved binding affinity.

CDR Randomization to Produce Higher Affinity Human scFv.

In an extension of simple site-directed mutagenesis, mutant antibodylibraries can be created where partial or entire CDRs are randomized(V_(L) CDR1 CDR2 and/or CDR3 and/or V_(H) CDR1, CDR2 and/or CDR3). Inone embodiment, each CDR is randomized in a separate library, using aknown antibody (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8),YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2,2C8, and/or UA8kappa) as a template. The CDR sequences of the highestaffinity mutants from each CDR library are combined to obtain anadditive increase in affinity. A similar approach has been used toincrease the affinity of human growth hormone (hGH) for the growthhormone receptor over 1500 fold from 3.4×10⁻¹⁰ to 9.0×10⁻¹³ M (Lowman etal. (1993) J. Mol. Biol., 234: 564-578).

V_(H) CDR3 often occupies the center of the binding pocket, and thusmutations in this region are likely to result in an increase in affinity(Clackson et al. (1995) Science, 267: 383-386). In one embodiment, V_(H)CDR3 residues are randomized (see, e.g., Schier et al. (1996) Gene, 169:147-155; Schier and Marks (1996) Human Antibodies and Hybridomas. 7:97-105, 1996; and Schier et al. (1996) J. Mol. Biol. 263: 551-567).

Other Antibody Modifications.

In one embodiment, partial antibody sequences derived from the YS5,YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8,YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappaantibody may be used to produce structurally and functionally relatedantibodies. For example, antibodies interact with target antigenspredominantly through amino acid residues that are located in the sixheavy and light chain complementarity determining regions (CDRs). Forthis reason, the amino acid sequences within CDRs are more diversebetween individual antibodies than sequences outside of CDRs. BecauseCDR sequences are responsible for most antibody-antigen interactions, itis possible to express recombinant antibodies that mimic the propertiesof specific naturally occurring antibodies by constructing expressionvectors that include CDR sequences from the specific naturally occurringantibody grafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann et al. (1998) Nature 332:323-327; Jones et al., (1986) Nature 321: 522-525; and Queen et al.(1989) Proc. Natl. Acad. Sci. USA, 86: 10029-10033). Such frameworksequences can be obtained from public DNA databases that includegermline antibody gene sequences.

Thus, one or more structural features of an anti-CD46 antibody of theinvention, such as the CDRs, can be used to create structurally relatedanti-CD46 antibodies that retain at least one functional property of,for example, the YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6,YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8,and/or UA8kappa antibody, e.g., binding and internalizing into prostatecancer cells.

In a particular embodiment, one or more YS5, YS5F, YS5v1D, SB1HGNY,YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11,3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa CDR regions (e.g. VH CDR1,and/or CDR2, and/or CDR3, and/or VL CDR1, and/or CDR2, and/or CDR3) iscombined recombinantly with known human framework regions and CDRs tocreate additional, recombinantly-engineered, anti-CD46 antibodies. Theheavy and light chain variable framework regions can be derived from thesame or different antibody sequences.

It is well known in the art that antibody heavy and light chain CDR3domains play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen (see, e.g., Hall etal. (1992) J. Immunol., 149: 1605-1612; Polymenis et al. (1994) J.Immunol., 152: 5318-5329; Jahn et al. (1995) Immunobiol., 193:400-419;Klimka et al. (2000) Brit. J. Cancer, 83: 252-260; Beiboer et al. (2000)J. Mol. Biol, 296: 833-849; Rader et al. (1998) Proc. Natl. Acad. Sci.USA, 95: 8910-8915; Barbas et al. (1994) J. Am. Chem. Soc., 116:2161-2162; Ditzel et al. (1996) J. Immunol., 157: 739-749). Accordingly,in certain embodiments, antibodies are generated that include the heavyand/or light chain CDR3s of the particular antibodies described herein(e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3,YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/orUA8kappa). Accordingly, in certain embodiments, antibodies are generatedthat include the heavy and/or light chain CDR1s of the particularantibodies described herein (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12,3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY,3G7NY, 3G7, SB2, 2C8, and/or UA8kappa). The antibodies can furtherinclude the other heavy and/or light chain CDRs of the antibodies of thepresent invention (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7,SB2, 2C8, and/or UA8kappa).

In certain embodiments the CDR1, 2, and/or 3 regions of the engineeredantibodies described above can comprise the exact amino acid sequence(s)as those disclosed herein (e.g., CDRs of YS5, YS5F, YS5v1D, SB1HGNY,YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11,3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa). However, the ordinarilyskilled artisan will appreciate that some deviation from the exact CDRsequences may be possible while still retaining the ability of theantibody to bind CD46 effectively (e.g., conservative amino acidsubstitutions). Accordingly, in another embodiment, the engineeredantibody may be composed of one or more CDRs that are, for example, 90%,95%, 98%, 99% or 99.5% identical to one or more CDRs of the YS5, YS5F,YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7,YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa antibody.

In another embodiment, one or more residues of a CDR may be altered tomodify binding to achieve a more favored on-rate of binding. Using thisstrategy, an antibody having ultra high binding affinity of, forexample, 10¹⁰ M⁻¹ or more, can be achieved. Affinity maturationtechniques, well known in the art and those described herein, can beused to alter the CDR region(s) followed by screening of the resultantbinding molecules for the desired change in binding. Accordingly, asCDR(s) are altered, changes in binding affinity as well asimmunogenicity can be monitored and scored such that an antibodyoptimized for the best combined binding and low immunogenicity areachieved.

In addition to, or instead of, modifications within the CDRs,modifications can also be made within one or more of the frameworkregions, FR1, FR2, FR3 and FR4, of the heavy and/or the light chainvariable regions of an antibody, so long as these modifications do noteliminate the binding affinity of the antibody.

In another embodiment, the antibody is further modified with respect toeffector function, so as to enhance the effectiveness of the antibody intreating cancer, for example. For example cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC) (see, e.g., Caron et al. (1992) J. Exp Med. 176:1191-1195; Shopes (1992) J. Immunol. 148: 2918-2922). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers (see, e.g., Wolff et al. (1993) CancerRes. 53:2560-2565). Alternatively, an antibody can be engineered whichhas dual Fc regions and may thereby have enhanced complement lysis andADCC capabilities (see, e.g., Stevenson et al. (1989) Anti-Cancer DrugDesign 3: 219-230).

Antibody Production.

In various embodiments antibodies described herein can be produced bychemical synthesis or can be recombinantly expressed.

Chemical Synthesis.

Using the sequence information provided herein, the CD46 specificantibodies described herein (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12,3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY,3G7NY, 3G7, SB2, 2C8, and/or UA8kappa), or variants thereof, can bechemically synthesized using well known methods of peptide synthesis.Solid phase synthesis in which the C-terminal amino acid of the sequenceis attached to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence is one preferred method forthe chemical synthesis of single chain antibodies. Techniques for solidphase synthesis are described by Barany and Merrifield, Solid PhasePeptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis,Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.,Merrifield et al. (1963) J. Am. Chem. Soc., 85: 2149-2156, and Stewartet al. (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill.

Recombinant Expression of Prostate Cancer-Specific Antibodies.

In certain embodiments, the CD46 specific antibodies described herein(e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3,YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/orUA8kappa), or variants thereof, are recombinantly expressed usingmethods well known to those of skill in the art. For example, using theYS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4,YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappasequence information provided herein, nucleic acids encoding the desiredantibody can be prepared according to a number of standard methods knownto those of skill in the art. The nucleic acids are transfected intohost cells that then express the desired antibody or a chain thereof.

Molecular cloning techniques to achieve these ends are known in the art.A wide variety of cloning and in vitro amplification methods aresuitable for the construction of recombinant nucleic acids. Examples ofthese techniques and instructions sufficient to direct persons of skillthrough many cloning exercises are found in Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology volume 152 AcademicPress, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Methods ofproducing recombinant immunoglobulins are also known in the art. See,Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. NatlAcad. Sci. USA 86: 10029-10033. In addition, detailed protocols for theexpression of antibodies are also provided by Liu et al. (2004) CancerRes. 64: 704-710, Poul et al. (2000) J. Mol. Biol. 301: 1149-1161, andthe like.

Creation of Other Antibody Forms.

Using the known and/or identified sequences (e.g. V_(H) and/or V_(L)sequences) of the single chain antibodies provided herein other antibodyforms can readily be created. Such forms include, but are not limited tomultivalent antibodies, full antibodies, scFv, (scFv′)₂, Fab, (Fab′)₂,chimeric antibodies, and the like.

Creation of Homodimers.

For example, to create (scFv′)₂ antibodies, two anti-CD46 antibodies arejoined, either through a linker (e.g., a carbon linker, a peptide, etc.)or through a disulfide bond between, for example, two cysteins. Thus,for example, to create disulfide linked scFv, a cysteine residue can beintroduced by site directed mutagenesis at the carboxy-terminus of theantibodies described herein.

An scFv can be expressed from this construct, purified by IMAC, andanalyzed by gel filtration. To produce (scFv′)₂ dimers, the cysteine isreduced by incubation with 1 mM 3-mercaptoethanol, and half of the scFvblocked by the addition of DTNB. Blocked and unblocked scFvs areincubated together to form (scFv′)₂ and the resulting material can beanalyzed by gel filtration. The affinity of the resulting dimmer can bedetermined using standard methods, e.g. by BIAcore.

In one illustrative embodiment, the (scFv′)₂ dimer is created by joiningthe scFv′ fragments through a linker, e.g., through a peptide linker.This can be accomplished by a wide variety of means well known to thoseof skill in the art. For example, one approach is described by Holligeret al. (1993) Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (see also WO94/13804).

It is noted that using the V_(H) and/or V_(L) sequences provided hereinFabs and (Fab′)₂ dimers can also readily be prepared. Fab is a lightchain joined to V_(H)-C_(H)1 by a disulfide bond and can readily becreated using standard methods known to those of skill in the art. TheF(ab)′₂ can be produced by dimerizing the Fab, e.g. as described abovefor the (scFv′)₂ dimer.

Chimeric Antibodies.

The antibodies contemplated herein also include “chimeric” antibodies inwhich a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see, e.g., U.S. Pat. No. 4,816,567; Morrison et al.(1984) Proc. Natl. Acad. Sci. 81: 6851-6855, etc.).

While the prototypic antibodies provided herein (e.g., YS5, YS5F,YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7,YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa) are fullyhuman antibodies, chimeric antibodies are contemplated, particularlywhen such antibodies are to be used in species other than humans (e.g.,in veterinary applications). Chimeric antibodies are antibodiescomprising portions from two different species (e.g. a human andnon-human portion). Typically, the antigen combining region (or variableregion) of a chimeric antibody is derived from a one species source andthe constant region of the chimeric antibody (which confers biologicaleffector function to the immunoglobulin) is derived from another source.A large number of methods of generating chimeric antibodies are wellknown to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,502,167,5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847, 5,292,867,5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235, 5,075,431, and4,975,369, and PCT application WO 91/0996).

In general, the procedures used to produce chimeric antibodies consistof the following steps (the order of some steps may be interchanged):(a) identifying and cloning the correct gene segment encoding theantigen binding portion of the antibody molecule; this gene segment(known as the VDJ, variable, diversity and joining regions for heavychains or VJ, variable, joining regions for light chains, or simply asthe V or variable region or V_(H) and V_(L) regions) may be in eitherthe cDNA or genomic form; (b) cloning the gene segments encoding thehuman constant region or desired part thereof; (c) ligating the variableregion to the constant region so that the complete chimeric antibody isencoded in a transcribable and translatable form; (d) ligating thisconstruct into a vector containing a selectable marker and gene controlregions such as promoters, enhancers and poly(A) addition signals; (e)amplifying this construct in a host cell (e.g., bacteria); (f)introducing the DNA into eukaryotic cells (transfection) most oftenmammalian lymphocytes; and culturing the host cell under conditionssuitable for expression of the chimeric antibody.

Antibodies of several distinct antigen binding specificities have beenmanipulated by these protocols to produce chimeric proteins (e.g.,anti-TNP: Boulianne et al. (1984) Nature, 312: 643) and anti-tumorantigens (see, e.g., Sahagan et al. (1986) J. Immunol., 137: 1066).Likewise several different effector functions have been achieved bylinking new sequences to those encoding the antigen binding region. Someof these include enzymes (Neuberger et al. (1984) Nature 312: 604),immunoglobulin constant regions from another species and constantregions of another immunoglobulin chain (Sharon et al. (1984) Nature309: 364; Tan et al., (1985) J. Immunol. 135: 3565-3567).

In certain embodiments, a recombinant DNA vector is used to transfect acell line that produces an anti-CD46 (e.g., a prostate cancer specific)antibody. The novel recombinant DNA vector contains a “replacement gene”to replace all or a portion of the gene encoding the immunoglobulinconstant region in the cell line (e.g., a replacement gene may encodeall or a portion of a constant region of a human immunoglobulin, aspecific immunoglobulin class, or an enzyme, a toxin, a biologicallyactive peptide, a growth factor, inhibitor, or a linker peptide tofacilitate conjugation to a drug, toxin, or other molecule, etc.), and a“target sequence” that allows for targeted homologous recombination withimmunoglobulin sequences within the antibody producing cell.

In another embodiment, a recombinant DNA vector is used to transfect acell line that produces an antibody having a desired effector function,(e.g., a constant region of a human immunoglobulin) in which case, thereplacement gene contained in the recombinant vector may encode all or aportion of a region of a prostate cancer specific antibody of thisinvention and the target sequence contained in the recombinant vectorallows for homologous recombination and targeted gene modificationwithin the antibody producing cell. In either embodiment, when only aportion of the variable or constant region is replaced, the resultingchimeric antibody can define the same antigen and/or have the sameeffector function yet be altered or improved so that the chimericantibody may demonstrate a greater antigen specificity, greater affinitybinding constant, increased effector function, or increased secretionand production by the transfected antibody producing cell line, etc.

Regardless of the embodiment practiced, the processes of selection forintegrated DNA (via a selectable marker), screening for chimericantibody production, and cell cloning, can be used to obtain a clone ofcells producing the chimeric antibody.

Thus, a piece of DNA that encodes a modification for a monoclonalantibody can be targeted directly to the site of the expressedimmunoglobulin gene within a B-cell or hybridoma cell line. DNAconstructs for any particular modification can be made to alter theprotein product of any monoclonal cell line or hybridoma. The level ofexpression of chimeric antibody should be higher when the gene is at itsnatural chromosomal location rather than at a random position. Detailedmethods for preparation of chimeric (humanized) antibodies can be foundin U.S. Pat. No. 5,482,856.

Intact Human Antibodies.

In another embodiment, this invention provides for intact, fully humananti-CD46 (e.g., prostate cancer specific) antibodies. Such antibodiescan readily be produced in a manner analogous to making chimeric humanantibodies. In this instance, instead of using a recognition functionderived, e.g. from a murine, the fully human recognition function (e.g.,VH and V_(L)) of the antibodies described herein is utilized.

Diabodies.

In certain embodiments, diabodies comprising one or more of the V_(H)and V_(L) domains described herein are contemplated. The term“diabodies” refers to antibody fragments typically having twoantigen-binding sites. The fragments typically comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (V_(H)-V_(L)). By using a linkerthat is too short to allow pairing between the two domains on the samechain, the domains are forced to pair with the complementary domains ofanother chain and create two antigen-binding sites. Diabodies aredescribed more fully in, for example, EP 404,097; WO 93/11161, andHolliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.

Unibodies.

In certain embodiments using the sequence information provided herein,the anti-CD46 antibodies can be constructed as unibodies. UniBody areantibody technology that produces a stable, smaller antibody format withan anticipated longer therapeutic window than certain small antibodyformats. In certain embodiments unibodies are produced from IgG4antibodies by eliminating the hinge region of the antibody. Unlike thefull size IgG4 antibody, the half molecule fragment is very stable andis termed a uniBody. Halving the IgG4 molecule leaves only one area onthe UniBody that can bind to a target. Methods of producing unibodiesare described in detail in PCT Publication WO2007/059782, which isincorporated herein by reference in its entirety (see, also, Kolfschotenet al. (2007) Science 317: 1554-1557).

Affibodies.

In certain embodiments the sequence information provided herein is usedto construct affibody molecules that bind CD46. Affibody molecules areclass of affinity proteins based on a 58-amino acid residue proteindomain, derived from one of the IgG-binding domains of staphylococcalprotein A. This three helix bundle domain has been used as a scaffoldfor the construction of combinatorial phagemid libraries, from whichaffibody variants that target the desired molecules can be selectedusing phage display technology (see, e.g., Nord et al. (1997) Nat.Biotechnol. 15: 772-777; Ronmark et al. (2002) Eur. J. Biochem., 269:2647-2655.). Details of Affibodies and methods of production are knownto those of skill (see, e.g., U.S. Pat. No. 5,831,012 which isincorporated herein by reference in its entirety).

It will be recognized that the antibodies described above can beprovided as whole intact antibodies (e.g., IgG), antibody fragments, orsingle chain antibodies, using methods well known to those of skill inthe art. In addition, while the antibody can be from essentially anymammalian species, to reduce immunogenicity, it is desirable to use anantibody that is of the species in which the antibody and/orimmunoconjugate is to be used. In other words, for use in a human, it isdesirable to use a human, humanized, or chimeric human antibody.

Measurement of Antibody/Polypeptide Binding Affinity.

As explained above, selection for increased avidity can involvesmeasuring the affinity of the antibody for the target antigen (e.g.,CD46, especially the epitope bound by one or more of YS5, YS5F, YS5v1D,SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10,YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa). Methods of makingsuch measurements are well known to those of skill in the art. Briefly,for example, the K_(d) of the antibody is determined from the kineticsof binding to, e.g. the target cell in a BIAcore, a biosensor based onsurface plasmon resonance. For this technique, the antigen or cell iscoupled to a derivatized sensor chip capable of detecting changes inmass. When antibody is passed over the sensor chip, antibody binds tothe antigen resulting in an increase in mass that is quantifiable.Measurement of the rate of association as a function of antibodyconcentration can be used to calculate the association rate constant(k_(on)). After the association phase, buffer is passed over the chipand the rate of dissociation of antibody (k_(off)) determined. K_(on) istypically measured in the range 1.0×10² to 5.0×10⁶ and k_(off) in therange 1.0×10⁻¹ to 1.0×10⁻⁶. The equilibrium constant K_(d) is oftencalculated as k_(off)/k_(on) and thus is typically measured in the range10⁻⁵ to 10⁻¹². Affinities measured in this manner correlate well withaffinities measured in solution by fluorescence quench titration.

Immunoconjguates Comprising YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7,SB2, 2C8, and/or UA8kappa or Other Anti-CD46 Antibodies.

The prototypical anti-CD46 antibodies (e.g., YS5, YS5F, YS5v1D, SB1HGNY,YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11,3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa) described hereinspecifically bind to and are internalized by prostate cancer cells andby other CD46 positive cancer cells. The antibodies can be used alone astherapeutics (e.g., to inhibit growth and/or proliferation of a prostatecancer cell) or they can be coupled to an effector formingimmunoconjugates that provide efficient and specific delivery of theeffector (e.g. cytotoxins, labels, radionuclides, ligands, antibodies,drugs, liposomes, nanoparticles, viral particles, cytokines, and thelike) to various cancer cells that express CD46 (e.g., isolated cells,metastatic cells, solid tumor cells, etc.).

Anti-CD46 immunoconjugates can be formed by conjugating the antibodiesor antigen binding portions thereof described herein to an effector(e.g., a detectable label, another therapeutic agent, etc.). Suitableagents include, for example, a cytotoxic or cytostatic agent (e.g., achemotherapeutic agent), a toxin (e.g. an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), and/ora radioactive isotope (i.e., a radioconjugate).

In certain embodiments, the effector comprises a detectable label.Suitable detectable labels include, but are not limited to radio-opaquelabels, nanoparticles, PET labels, MRI labels, radioactive labels, andthe like. Among the radionuclides and useful in various embodiments ofthe present invention, gamma-emitters, positron-emitters, x-ray emittersand fluorescence-emitters are suitable for localization, diagnosisand/or staging, and/or therapy, while beta and alpha-emitters andelectron and neutron-capturing agents, such as boron and uranium, alsocan be used for therapy.

The detectable labels can be used in conjunction with an externaldetector and/or an internal detector and provide a means of effectivelylocalizing and/or visualizing prostate cancer cells. Suchdetection/visualization can be useful in various contexts including, butnot limited to pre-operative and intraoperative settings. Thus, incertain embodiment this invention relates to a method ofintraoperatively detecting and prostate cancers in the body of a mammal.These methods typically involve administering to the mammal acomposition comprising, in a quantity sufficient for detection by adetector (e.g. a gamma detecting probe), an prostate cancer specificantibody labeled with a detectable label (e.g. antibodies of thisinvention labeled with a radioisotope, e.g. ¹⁶¹Tb, ¹²³I, ¹²⁵I, and thelike), and, after allowing the active substance to be taken up by thetarget tissue, and preferably after blood clearance of the label,subjecting the mammal to a radioimmunodetection technique in therelevant area of the body, e.g. by using a gamma detecting probe.

In certain embodiments the label-bound antibody can be used in thetechnique of radioguided surgery, wherein relevant tissues in the bodyof a subject can be detected and located intraoperatively by means of adetector, e.g. a gamma detecting probe. The surgeon can,intraoperatively, use this probe to find the tissues in which uptake ofthe compound labeled with a radioisotope, that is, e.g. a low-energygamma photon emitter, has taken place. In certain embodiments suchmethods are particularly useful in localizing and removing secondarycancers produced by metastatic cells from a primary tumor.

In addition to detectable labels, certain preferred effectors include,but are not limited to cytotoxins (e.g. Pseudomonas exotoxin, ricin,abrin, Diphtheria toxin, and the like), or cytotoxic drugs or prodrugs,in which case the chimeric molecule may act as a potent cell-killingagent specifically targeting the cytotoxin to prostate cancer cells.

In still other embodiments, the effector can include a liposomeencapsulating a drug (e.g. an anti-cancer drug such as abraxane,doxorubicin, pamidronate disodium, anastrozole, exemestane,cyclophosphamide, epirubicin, toremifene, letrozole, trastuzumab,megestroltamoxifen, paclitaxel, docetaxel, capecitabine, goserelinacetate, zoledronic acid, vinblastine, etc.), an antigen that stimulatesrecognition of the bound cell by components of the immune system, anantibody that specifically binds immune system components and directsthem to the prostate cancer, and the like.

Illustrative Effectors.

Imaging Compositions.

In certain embodiments, the anti-CD46 immunoconjugates can be used todirect detectable labels to a tumor site. This can facilitate tumordetection and/or localization. It can be effective for detecting primarytumors, or, in certain embodiments, secondary tumors produced by, e.g.,prostate metastatic cells. In certain embodiments, the effectorcomponent of the immunoconjugate comprises a “radio-opaque” label, e.g.a label that can be easily visualized using x-rays. Radio-opaquematerials are well known to those of skill in the art. The most commonradio-opaque materials include iodide, bromide or barium salts. Otherradiopaque materials are also known and include, but are not limited to,organic bismuth derivatives (see, e.g., U.S. Pat. No. 5,939,045),radio-opaque polyurethanes (see, e.g., U.S. Pat. No. 5,346,981),organobismuth composites (see, e.g., U.S. Pat. No. 5,256,334),radio-opaque barium polymer complexes (see, e.g., U.S. Pat. No.4,866,132), and the like.

The anti-CD46 antibodies described herein can be coupled directly to theradio-opaque moiety or they can be attached to a “package” (e.g., achelate, a liposome, a polymer microbead, a nanoparticle, etc.)carrying, containing, or comprising the radio-opaque material, e.g., asdescribed below.

In addition to radio-opaque labels, other labels are also suitable foruse. Detectable labels suitable for use in immunoconjugates include anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe include magnetic beads (e.g., DYNABEADS™), fluorescent dyes (e.g.,fluorescein isothiocyanate, texas red, rhodamine, green fluorescentprotein, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C or ³²P),enzymes (e.g., horse radish peroxidase, alkaline phosphatase and otherscommonly used in an ELISA), and colorimetric labels such as colloidalgold or colored glass or plastic (e.g. polystyrene, polypropylene,latex, etc.) beads, nanoparticles, quantum dots, and the like.

In certain embodiments, suitable radiolabels include, but are notlimited to, ⁹⁹Te, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ^(113m)In, ⁹⁷Ru, ⁶²Cu,641Cu, ⁵²Fe, ^(52m)Mn, ⁵¹Cr, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er,¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm,¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh,and ¹¹¹Ag.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, certain radiolabels may be detected usingphotographic film, scintillation detectors, PET imaging, MRI, and thelike. Fluorescent markers can be detected using a photodetector todetect emitted illumination. Enzymatic labels are typically detected byproviding the enzyme with a substrate and detecting the reaction productproduced by the action of the enzyme on the substrate, and colorimetriclabels are detected by simply visualizing the colored label.

Radiosensitizers.

In another embodiment, the effector can comprise a radiosensitizer thatenhances the cytotoxic effect of ionizing radiation (e.g., such as mightbe produced by ⁶⁰Co or an x-ray source) on a cell. Numerousradiosensitizing agents are known and include, but are not limited tobenzoporphyrin derivative compounds (see, e.g., U.S. Pat. No.5,945,439), 1,2,4-benzotriazine oxides (see, e.g., U.S. Pat. No.5,849,738), compounds containing certain diamines (see, e.g., U.S. Pat.No. 5,700,825), BCNT (see, e.g., U.S. Pat. No. 5,872,107),radiosensitizing nitrobenzoic acid amide derivatives (see, e.g., U.S.Pat. No. 4,474,814), various heterocyclic derivatives (see, e.g., U.S.Pat. No. 5,064,849), platinum complexes (see, e.g., U.S. Pat. No.4,921,963), and the like.

Alpha Emitters.

In certain embodiments, the effector can include an alpha emitter, i.e.a radioactive isotope that emits alpha particles. Alpha-emitters haverecently been shown to be effective in the treatment of cancer (see,e.g., McDevitt et al. (2001) Science 294:1537-1540; Ballangrud et al.(2001) Cancer Res. 61: 2008-2014; Borchardt et al. (2003) Cancer Res.63: 5084-50). Suitable alpha emitters include, but are not limited toBi, ²¹³Bi, ²¹¹At, and the like.

Chelates

Many of the pharmaceuticals and/or radiolabels described herein can beprovided as a chelate. The chelating molecule is typically coupled to amolecule (e.g. biotin, avidin, streptavidin, etc.) that specificallybinds an epitope tag attached to an anti-CD46 antibody (e.g., Y55, YS5F,YS5v1D, SB1HGNY, Y512, 3G7RY (aka 3G8), Y56, YS1, Y53, Y54, Y58, Y57,Y59, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa) describedherein.

Chelating groups are well known to those of skill in the art. In certainembodiments, chelating groups are derived from ethylene diaminetetra-acetic acid (EDTA), diethylene triamine penta-acetic acid (DTPA),cyclohexyl 1,2-diamine tetra-acetic acid (CDTA),ethyleneglycol-O,O′-bis(2-aminoethyl)-N,N,N′,N′-tetra-acetic acid(EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid(HBED), triethylene tetramine hexa-acetic acid (TTHA),1,4,7,10-tetraazacyclododecane-N,N′—,N″,N′″-tetra-acetic acid (DOTA),hydroxyethyldiamine triacetic acid (HEDTA),1,4,8,11-tetra-azacyclotetradecane-N,N′,N″,N′″-tetra-acetic acid (TETA),substituted DTPA, substituted EDTA, and the like.

Examples of certain preferred chelators include unsubstituted or,substituted 2-iminothiolanes and 2-iminothiacyclohexanes, in particular2-imino-4-mercaptomethylthiolane.

One chelating agent, 1,4,7,10-tetraazacyclododecane-N, N, N″,N′″-tetraacetic acid (DOTA), is of particular interest because of itsability to chelate a number of diagnostically and therapeuticallyimportant metals, such as radionuclides and radiolabels.

Conjugates of DOTA and proteins such as antibodies have been described.For example, U.S. Pat. No. 5,428,156 teaches a method for conjugatingDOTA to antibodies and antibody fragments. To make these conjugates, onecarboxylic acid group of DOTA is converted to an active ester which canreact with an amine or sulfhydryl group on the antibody or antibodyfragment. Lewis et al. (1994) Bioconjugate Chem. 5: 565-576, describes asimilar method wherein one carboxyl group of DOTA is converted to anactive ester, and the activated DOTA is mixed with an antibody, linkingthe antibody to DOTA via the epsilon-amino group of a lysine residue ofthe antibody, thereby converting one carboxyl group of DOTA to an amidemoiety.

In certain embodiments the chelating agent can be coupled, directly orthrough a linker, to an epitope tag or to a moiety that binds an epitopetag. Conjugates of DOTA and biotin have been described (see, e.g., Su(1995) J. Nucl. Med., 36 (5 Suppl):154P, which discloses the linkage ofDOTA to biotin via available amino side chain biotin derivatives such asDOTA-LC-biotin or DOTA-benzyl-4-(6-amino-caproamide)-biotin). Yau etal., WO 95/15335, disclose a method of producing nitro-benzyl-DOTAcompounds that can be conjugated to biotin. The method comprises acyclization reaction via transient projection of a hydroxy group;tosylation of an amine; deprotection of the transiently protectedhydroxy group; tosylation of the deprotected hydroxy group; andintramolecular tosylate cyclization. Wu et al. (1992) Nucl. Med. Biol.,19(2): 239-244 discloses a synthesis of macrocylic chelating agents forradiolabeling proteins with ¹¹¹IN and ⁹⁰Y. Wu et al. makes a labeledDOTA-biotin conjugate to study the stability and biodistribution ofconjugates with avidin, a model protein for studies. This conjugate wasmade using a biotin hydrazide which contained a free amino group toreact with an in situ generated activated DOTA derivative.

Cytotoxins/Cytostatic Agents.

The anti-CD46 antibodies described herein (e.g., YS5, YS5F, YS5v1D,SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10,YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa) can be used todeliver a variety of cytotoxic and/or cytostatic drugs includingtherapeutic drugs, a compound emitting radiation, cytotoxic molecules ofplant, fungal, or bacterial origin, biological proteins, and mixturesthereof. In certain embodiments the cytotoxic drugs can compriseintracellularly acting cytotoxic drugs that are, e.g., small organicmolecules, cytotoxic proteins or peptides, radiation emitters,including, for example, short-range, high-energy α-emitters as describedabove, and the like.

Accordingly, in certain embodiments, the anti-CD46 antibody is attachedto a cytotoxic/cytostatic drug. In various embodiments the drugs beingused to construct ADCs include, but are not limited to microtubuleinhibitors and DNA-damaging agents, polymerase inhibitors (e.g., thepolymerase II inhibitor, α-amanitin), and the like. In certainembodiments the antibody is conjugated to the drug directly or through alinker, while in other embodiments, the antibody is conjugated to a drugcarrier (e.g., a liposome containing the drug, a polymeric drug carrier,a nanoparticle drug carrier, a lipid drug carrier, a dendrimeric drugcarrier, and the like).

In certain embodiments the drug comprises a tubulin inhibitor,including, but not limited to auristatin, Dolastatin-10, syntheticderivatives of the natural product Dolastatin-10, and maytansine or amaytansine derivative.

In certain embodiments the drug comprises an auristatin. In certainembodiments the auristatin is selected from the group consisting of:Auristatin E (AE), Monomethylauristatin E (MMAE), Monomethylauristatin F(MMAF), vcMMAE, and vcMMAF.

In certain embodiments the drug comprises a maytansine. Illustrativemaytansines include, but are not limited to, Mertansine (DM1); and ananalogue of maytansine such as DM3 or DM4.

In certain embodiments the drug comprises a DNA interacting agent. Incertain embodiments the DNA interacting agent includes, but is notlimited to calicheamicins, duocarmycins, pyrrolobenzodiazepines (PBDs),and the like.

In one illustrative, but non-limiting embodiment, the drug comprises acalicheamicin. Calicheamicins target DNA and cause strand scission. Incertain embodiments the drug comprises calicheamicin or a calicheamicinanalog. Calicheamicin analogs are described in U.S. Pat. No. 5,264,586,which is incorporated herein by reference for the calicheamicin analogsdescribed therein.

In another illustrative, but non-limiting embodiment, the drug comprisesa duocarmycin. Duocarmycins are DNA damaging agents able to exert theirmode of action at any phase in the cellular cycle. Agents that are partof this class of duocarmycins typically have potency in the lowpicomolar range. Illustrative duocarmyhcins (e.g., duocarmycinanalogues) that can be used as effectors in the chimeric constructscontemplated herein include, but are not limited to duocarmycin A,duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2,duocarmycin D, duocarmycin SA, Cyclopropylbenzoindole duocarmycin(CC-1065), Centanamycin, Rachelmycin, Adozelesin, Bizelesin, Carzelesin,and the like.

In another illustrative, but non-limiting embodiment, the drug comprisesa pyrrolobenzodiazepine. In certain embodiments the drug comprises asynthetic derivative of two pyrrolobenzodiazepines linked by a flexiblepolymethylene tether. Pyrrolobenzodiazepines (PBDs) and PBD dimers aredescribed in U.S. Pat. No. 7,528,126 B2, which is incorporated herein byreference for the Pyrrolobenzodiazepines and PBD dimers describedtherein. In certain embodiments the pyrrolobenzodiazepine is selectedfrom the group consisting of: Anthramycin (and dimers thereof),Mazethramycin (and dimers thereof), Tomaymycin (and dimers thereof),Prothracarcin (and dimers thereof), Chicamycin (and dimers thereof),Neothramycin A (and dimers thereof), Neothramycin B (and dimersthereof), DC-81 (and dimers thereof), Sibiromycin (and dimers thereof),Porothramycin A (and dimers thereof), Porothramycin B (and dimersthereof), Sibanomycin (and dimers thereof), Abbeymycin (and dimersthereof), SG2000, and SG2285.

In certain embodiments the drug comprise a polymerase inhibitor,including, but not limited to polymerase II inhibitors such asα-amanitin, and poly(ADP-ribose) polymerase (PARP) inhibitors.Illustratiev PARP inhibitors include, but are not limited to Iniparib(BSI 201), Talazoparib (BMN-673), Olaparib (AZD-2281), Olaparib,Rucaparib (AG014699, PF-01367338), Veliparib (ABT-888), CEP 9722, MK4827, BGB-290, 3-aminobenzamide, and the like.

In certain embodiments the cytotoxic/cytostatic agent comprises aprotein or peptide toxin or fragment thereof. Enzymatically activetoxins and fragments thereof are exemplified by diphtheria toxin Afragment, nonbinding active fragments of diphtheria toxin, exotoxin A(from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin Achain, α-sacrin, certain Aleurites fordii proteins, certain Dianthinproteins, Phytolacca americana proteins (PAP, PAPII and PAP-S), Morodicacharantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor,gelonin, mitogillin, restrictocin, phenomycin, enomycin, and thetricothecenes, for example. A variety of radionuclides are available forthe production of radioconjugated antibodies. Examples include, but arenot limited to ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, ¹⁸⁶Re, and the like.

In certain embodiments the cytotoxins can include, but are not limitedto Pseudomonas exotoxins, Diphtheria toxins, ricin, abrin andderivatives thereof. Pseudomonas exotoxin A (PE) is an extremely activemonomeric protein (molecular weight 66 kD), secreted by Pseudomonasaeruginosa, which inhibits protein synthesis in eukaryotic cells throughthe inactivation of elongation factor 2 (EF-2) by catalyzing itsADP-ribosylation (catalyzing the transfer of the ADP ribosyl moiety ofoxidized NAD onto EF-2).

The toxin contains three structural domains that act in concert to causecytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding.Domain II (amino acids 253-364) is responsible for translocation intothe cytosol and domain III (amino acids 400-613) mediates ADPribosylation of elongation factor 2, which inactivates the protein andcauses cell death. The function of domain Ib (amino acids 365-399)remains undefined, although a large part of it, amino acids 365-380, canbe deleted without loss of cytotoxicity. See Siegall et al. (1989) J.Biol. Chem. 264: 14256-14261.

In certain embodiments the antibody is attached to a preferred moleculein which domain Ia (amino acids 1 through 252) is deleted and aminoacids 365 to 380 have been deleted from domain Ib. In certainembodiments all of domain Ib and a portion of domain II (amino acids 350to 394) can be deleted, particularly if the deleted sequences arereplaced with a linking peptide.

In addition, the PE and other cytotoxic proteins can be further modifiedusing site-directed mutagenesis or other techniques known in the art, toalter the molecule for a particular desired application. For example,means to alter the PE molecule in a manner that does not substantiallyaffect the functional advantages provided by the PE molecules describedhere can also be used and such resulting molecules are intended to becovered herein.

Methods of cloning genes encoding PE fused to various ligands are wellknown to those of skill in the art (see, e.g., Siegall et al. (1989)FASEB J., 3: 2647-2652; and Chaudhary et al. (1987) Proc. Natl. Acad.Sci. USA, 84: 4538-4542).

Like PE, diphtheria toxin (DT) kills cells by ADP-ribosylatingelongation factor 2 thereby inhibiting protein synthesis. Diphtheriatoxin, however, is divided into two chains, A and B, linked by adisulfide bridge. In contrast to PE, chain B of DT, which is on thecarboxyl end, is responsible for receptor binding and chain A, which ispresent on the amino end, contains the enzymatic activity (Uchida et al.(1972) Science, 175: 901-903; Uchida et al. (1973) J. Biol. Chem., 248:3838-3844).

In certain embodiments, the antibody-Diphtheria toxin immunoconjugatesof this invention have the native receptor-binding domain removed bytruncation of the Diphtheria toxin B chain. One illustrative modifiedDipththeria toxin is DT388, a DT in which the carboxyl terminal sequencebeginning at residue 389 is removed (see, e.g., Chaudhary et al. (1991)Bioch. Biophys. Res. Comm., 180: 545-551). Like the PE chimericcytotoxins, the DT molecules can be chemically conjugated to theprostate cancer specific antibody, but, in certain preferredembodiments, the antibody will be fused to the Diphtheria toxin byrecombinant means (see, e.g., Williams et al. (1990) J. Biol. Chem. 265:11885-11889).

Viral Particles.

In certain embodiments, the effector comprises a viral particle (e.g., afilamentous phage, an adeno-associated virus (AAV), a lentivirus, andthe like). The antibody can be conjugated to the viral particle and/orcan be expressed on the surface of the viral particle (e.g. afilamentous phage). The viral particle can additionally include anucleic acid that is to be delivered to the target (e.g., prostatecancer) cell. The use of viral particles to deliver nucleic acids tocells is described in detail in WO 99/55720, U.S. Pat. Nos. 6,670,188,6,642,051, and 6,669,936.

Other Therapeutic Moieties.

Other suitable effector molecules include pharmacological agents orencapsulation systems containing various pharmacological agents. Thus,in various embodiments, it is recognized that the targeting molecule(e.g., the targeting antibody) can be attached directly or through alinker to a drug that is to be delivered directly to the tumor.

Such drugs are well known to those of skill in the art and include, butare not limited to, anti-cancer antibodies (e.g., HERCEPTIN®),antimetabolites, alkylating agents, topoisomerase inhibitors,microtubule targeting agents, kinase inhibitors, protein synthesisinhibitors, somatostatin analogs, glucocorticoids, aromatose inhibitors,mTOR inhibitors, protein Kinase B (PKB) inhibitors,phosphatidylinositol, 3-Kinase (PI3K) Inhibitors, cyclin dependentkinase inhibitors, anti-TRAIL molecules, MEK inhibitors, and the like.In certain embodiments the anti-cancer compounds include, but are notlimited to flourouracil (5-FU), capecitabine/XELODA,5-Trifluoromethyl-2′-deoxyuridine, methotrexate sodium,raltitrexed/Tomudex, pemetrexed/Alimta®, cytosine Arabinoside(Cytarabine, Ara-C)/Thioguanine, 6-mercaptopurine (Mercaptopurine,6-MP), azathioprine/Azasan, 6-thioguanine (6-TG)/Purinethol (TEVA),pentostatin/Nipent, fludarabine phosphate/Fludara®, cladribine (2-CdA,2-chlorodeoxyadenosine)/Leustatin, floxuridine (5-fluoro-2)/FUDR(Hospira, Inc.), ribonucleotide Reductase Inhibitor (RNR),cyclophosphamide/Cytoxan (BMS), neosar, ifosfamide/Mitoxana, thiotepa,BCNU-1,3-bis(2-chloroethyl)-1-nitosourea,1,-(2-chloroethyl)-3-cyclohexyl-lnitrosourea, methyl CCNU,hexamethylmelamine, busulfan/Myleran, procarbazine HCL/Matulane,dacarbazine (DTIC), chlorambucil/Leukaran®, melphalan/Alkeran, cisplatin(Cisplatinum, CDDP)/Platinol, carboplatin/Paraplatin,oxaliplatin/Eloxitan, bendamustine, carmustine, chloromethine,dacarbazine (DTIC), fotemustine, lomustine, mannosulfan, nedaplatin,nimustine, prednimustine, ranimustine, satraplatin, semustine,streptozocin, temozolomide, treosulfan, triaziquone, triethylenemelamine, thioTEPA, triplatin tetranitrate, trofosfamide, uramustine,doxorubicin HCL/Doxil, daunorubicin citrate/Daunoxome®, mitoxantroneHCL/Novantrone, actinomycin D, etoposide/Vepesid, topotecanHCL/Hycamtin, teniposide (VM-26), irinotecan HCL(CPT-11)/Camptosar®,camptothecin, Belotecan, rubitecan, vincristine, vinblastine sulfate,vinorelbine tartrate, vindesine sulphate, paclitaxel/Taxol,docetaxel/Taxotere, nanoparticle paclitaxel, abraxane, ixabepilone,larotaxel, ortataxel, tesetaxel, vinflunine, and the like. In certainembodiments the anti-cancer drug(s) comprise one or more drugs selectedfrom the group consisting of carboplatin (e.g., PARAPLATIN®), Cisplatin(e.g., PLATINOL®, PLATINOL-AQ®), Cyclophosphamide (e.g., CYTOXAN®,NEOSAR®), Docetaxel (e.g., TAXOTERE®), Doxorubicin (e.g., ADRIAMYCIN®),Erlotinib (e.g., TARCEVA®), Etoposide (e.g., VEPESID®), Fluorouracil(e.g., 5-FU®), Gemcitabine (e.g., GEMZAR®), imatinib mesylate (e.g.,GLEEVEC®), Irinotecan (e.g., CAMPTOSAR®), Methotrexate (e.g., FOLEX®,MEXATE®, AMETHOPTERIN®), Paclitaxel (e.g., TAXOL®, ABRAXANE®), Sorafinib(e.g., NEXAVAR®), Sunitinib (e.g., SUTENT®), Topotecan (e.g.,HYCAMTIN®), Vinblastine (e.g., VELBAN®), Vincristine (e.g., ONCOVIN®,VINCASAR PFS®). In certain embodiments the anti-cancer drug comprisesone or more drugs selected from the group consisting of retinoic acid, aretinoic acid derivative, doxirubicin, vinblastine, vincristine,cyclophosphamide, ifosfamide, cisplatin, 5-fluorouracil, a camptothecinderivative, interferon, tamoxifen, and taxol. In certain embodiments theanti-cancer compound is selected from the group consisting of abraxane,doxorubicin, pamidronate disodium, anastrozole, exemestane,cyclophosphamide, epirubicin, toremifene, letrozole, trastuzumab,megestroltamoxifen, paclitaxel, docetaxel, capecitabine, goserelinacetate, zoledronic acid, vinblastine, etc.), an antisense molecule, anSiRNA, and the like.

Alternatively, the effector molecule can comprise an encapsulationsystem, such as a viral capsid, a liposome, or micelle that contains atherapeutic composition such as a drug, a nucleic acid (e.g. anantisense nucleic acid or another nucleic acid to be delivered to thecell), or another therapeutic moiety that is preferably shielded fromdirect exposure to the circulatory system. Means of preparing liposomesattached to antibodies are well known to those of skill in the art (see,e.g., U.S. Pat. No. 4,957,735, Connor et al. (1985) Pharm. Ther., 28:341-365, and the like).

B) Attachment of the Antibody to the Effector.

One of skill will appreciate that the anti-CD46 antibodies describedherein (e.g., YS5, YS5F, YS5v1D, SB1HGNY, YS12, 3G7RY (aka 3G8), YS6,YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8,and/or UA8kappa) and the effector molecule(s) can be joined together inany order. Thus, where antibody is a single chain polypeptide, theeffector molecule can be joined to either the amino or carboxy terminiof the targeting molecule. The antibody can also be joined to aninternal region of the effector molecule, or conversely, the effectormolecule can be joined to an internal location of the antibody, as longas the attachment does not interfere with the respective activities ofthe molecules.

The antibody and the effector can be attached by any of a number ofmeans well known to those of skill in the art. Typically the effector isconjugated, either directly or through a linker (spacer), to theantibody. However, in certain embodiments, where both the effectormolecule is or comprises a polypeptide it is preferable to recombinantlyexpress the chimeric molecule as a single-chain fusion protein.

Conjugation of the Effector Molecule to the Antibody.

In one embodiment, the CD46 specific antibody is chemically conjugatedto the effector molecule (e.g., a cytotoxin, a label, a ligand, a drug,a liposome, etc.). Means of chemically conjugating molecules are wellknown to those of skill.

The procedure for attaching an effector to an antibody will varyaccording to the chemical structure of the effector and/or antibody.Polypeptides typically contain variety of functional groups; e.g.,carboxylic acid (COOH) or free amine (—NH₂) groups, that are availablefor reaction with a suitable functional group on an effector molecule tobind the effector thereto.

Alternatively, the antibody and/or the effector can be derivatized toexpose or attach additional reactive functional groups. Thederivatization can involve attachment of any of a number of linkermolecules such as those available from Pierce Chemical Company, RockfordIll.

A “linker”, as used herein, is a molecule that is used to join thetargeting molecule to the effector molecule. The linker is capable offorming covalent bonds to both the targeting molecule and to theeffector molecule. Suitable linkers are well known to those of skill inthe art and include, but are not limited to, straight or branched-chaincarbon linkers, heterocyclic carbon linkers, or peptide linkers. Wherethe targeting molecule and the effector molecule are polypeptides, thelinkers may be joined to the constituent amino acids through their sidegroups (e.g., through a disulfide linkage to cysteine). However, in apreferred embodiment, the linkers will be joined to the alpha carbonamino or carboxyl groups of the terminal amino acids.

The immunoconjugates can be made using a variety of bifunctional proteincoupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters(such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as tolyene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Forexample, a ricin immunotoxin can be prepared as described in Vitetta etal., Science 238: 1098 (1987). Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is an illustrative, but non-limiting, chelating agent forconjugation of, e.g., a radionucleotide to the antibody (see, e.g.,WO1994/011026 (PCT/US1993/010953)).

In certain embodiments conjugation of effectors (e.g., drugs, liposomes,etc.). or linkers attached to effectors, to an antibody takes place atsolvent accessible reactive amino acids such as lysines or cysteinesthat can be derived from the reduction of inter-chain disulfide bonds inthe antibody. In certain embodiments cysteine conjugation can occurafter reduction of four inter-chain disulfide bonds.

In certain embodiments site-specific conjugation, in which a knownnumber of linker-drugs are consistently conjugated to defined sites inthe antibody can be performed to produce a highly homogenous construct.Drug-to-antibody ratio (DAR) can precisely controlled and can betailored to various linker-drugs, producing, for example, either 2- or4-DAR site-specific ADCs.

A number of methods are known to achieve sites-specific conjugation. Forexample, the amino acid cysteine contains a reactive thiol group thatserves essential roles in the structure and function of many proteins.Conjugation of thio-reactive probes to proteins through cysteineresidues has long been a method for protein labeling, and it has alsobeen applied to the generation of antibody drug conjugates (ADCs). Incertain illustrative, but non-limiting embodiments, this processinvolves partial reduction of existing disulfide bonds (e.g., interchaindisulfide bonds).

In certain embodiments to maintain disulfide bonds, cysteine residuescan be engineered into proteins. The success of using introducedcysteine residues for site-specific conjugation relies on the ability toselect proper sites in which cysteine-substitution does not alterprotein structure or function. To accomplish this, the Phage Elisa forSelection of Reactive Thiols (PHESELECTOR) was developed by introducingreactive cysteine residues into an antibody-Fab (trastuzumab-Fab 4D5) atvarious sites, displaying the Fab on phage, and screening to identifyreactive cysteines that do not interfere with antigen binding (see,e.g., Junutula et al. (2008) J. Immunol. Meth. 332: 41-52).

The PHESELECTOR approach has been demonstrated to be efficient andspecific, especially compared with conventional cysteine conjugation. Ithas been demonstrated that the optimal sites for cysteine found using,e.g., an antibody fragment (e.g., Fab) and the PHESELECTOR method canalso be applied to full-length antibodies, and data indicate that thesesites work well for site-specific conjugation to other mAbs (see, e.g.,Boswell et al. (2011) Bioconjug. Chem. 22: 1994-2004; Boswell et al.(2012) Soc. Nuclear Med. 53: 1454-1461; Shen et al. (2012) Nat.Biotechnol. 30:184-189).

Another illustrative, but non-limiting strategy for site-specificconjugation centers on the insertion of amino acids with bio-orthogonalreactive handles such as the amino acid selenocysteine and the unnaturalamino acid, acetylphenylalanine (pAcPhe). Two methods have beendeveloped to employ these amino acids and both utilize stop codons.However, one method incorporates selenocysteine (Sec) by pairing theopal stop codon, UGA, with a Sec insertion sequence and the other methodincorporates acetylphenylalanine at the amber stop codon, UAG, using atRNA/aminoacyltRNA synthetase pair. Selenocysteine, employed by thefirst method, is very similar to the amino acid, cysteine, but containsa selenium atom in place of the sulfur atom. The selenolate group is amore reactive nucleophile than the thiolate counterpart, rendering itamenable to conjugation with electrophilic compounds under conditions inwhich selenocysteine is selectively activated. There are approximately25 known selenium-containing proteins in mammals, including proteinssuch as glutathione peroxidases and thioreductases (Kryukov et al.92003) Science, 300: 1439-1443). Under normal conditions, UGA codes fortranscriptional termination; however, in the presence of a Sec insertionsequence (SECIS) located in the 3′ UTR of Sec containing proteins,termination is prevented by the formation of an mRNA secondary structureand Sec is inserted at the UGA codon (Caban and Copeland (2006) CellMol. Life Sci. 63: 73-81). Sec insertion can be engineered into non-Seccoding genes by insertion of the UGA codon and a SECIS at the 3′ end ofthe gene. This technique has been used, inter alia, in the Sec labelingand subsequent site-specific conjugation of mAbs (see, e.g., Hofer etal. (2009) Biochem. 48: 12047-12057).

Still another illustrative method for site-specific conjugation utilizesthe unnatural amino acid, p-acetylphenylalanine (pAcPhe). pAcPhecontains a keto group that can be selectively conjugated to a drugcontaining an alkoxy-amine through an oxime ligation. To incorporatepAcPhe into an antibody, the amber stop codon is substituted into theantibody at the desired location. The antibody cDNA is then co-expressedwith an amber suppressor tRNA and the properly paired mutant tRNAsythetase. The tRNA sythetase loads pAcPhe onto the amber tRNA and thuspAcPhe is incorporated into the antibody at the amber site UAG (see,e.g., Liu et al. 92007) Nat. Meth. 4: 239-244; Wang et al. (2003) Proc.Natl. Acad. Sci. USA, 100: 56-61; Axup (2012) Proc. Natl. Acad. Sci.USA, 109: 16101-16116).

In addition to pAcPhe, other unnatural amino acids are exploited for usein site-specific conjugation using similar processes involving matchingtRNA/aminoacyltRNA synthetase pairs (see, e.g., Young (2002) J. Mol.Biol. 395: 361-374; Kiick et al. (2002) Proc. Natl. Acad. Sci. USA, 99:19-24).

In various embodiments the use of enzymes to catalyze bond formation canbe exploited for use in site-specific conjugation. For example, theglycotransferase platform uses a mutant glycotransferase to attach achemically active sugar moiety to a glycosylation site on an antibody.Molecules of choice can then be conjugated to the chemical handle on thesugar moiety. In another illustrative, but non-limiting approachtransglutaminase is used to form a bond between an amine group on thelinker/drug and an engineered glutamine residue on the antibody.

Glycotransferases are a large family of proteins involved in thesynthesis of oligosaccharides and are responsible for the transfer of asugar residue from an activated sugar nucleotide to a sugar acceptor orglycoprotein/lipid. The structures of several glycotransferases areknown and reveal that sugar donor specificity is determined by a fewamino acids in the catalytic pocket (Qasba et al. (2005) Trends Biochem.Sci. 30: 53-62), Using this knowledge, residues have been mutated in thepocket of the glycotransferase, e.g., B4Gal-T1, to broaden donorspecificity and allow the transfer of the chemically reactive sugarresidue, 2-keto-Gal (see, e.g., Ramakrishnan et al. (2002) J. Biol.Chem. 277: 20833-20839). This technology allows for the ability totransfer a chemically reactive sugar to any lipid or protein containinga glycosylation site. Human IgG antibodies contain an N-glycosylationsite at the conserved Asn-297 of the Fc fragment. The glycans attachedto this site are generally complex, but can be degalactosylated down toGO, onto which a mutant glycotransferase is capable of transferringC2-keto-Gal with high efficiency (see, e.g., Boeggeman et al. (2009)Bioconjug. Chem. 20: 1228-1236). The active chemical handle of C2-ketoGal can then be coupled to biomolecules with an orthogonal reactivegroup. This approach has been used successfully for the site-specificconjugation of the anti-Her2 antibody, trastuzumab, with Alexa Fluor 488aminooxyacetamide and is a viable technique for sitespecific ADCgeneration (Id.).

The second platform utilizes transglutaminase to catalyze the formationof a covalent bond between a free amine group and a glutamine sidechain. Transglutaminase from Streptoverticillium mobaraense (mTG) iscommercially available and has been used extensively as a proteincrosslinking agent (see, e.g., Yokoyama et al. (2004) Appl. Microbiol.Biotechnol. 64: 447-454). mTG does not recognize any of the naturaloccurring glutamine residues in the Fc region of glycosylatedantibodies, but does recognize a “glutamine tag” that can be engineeredinto an antibody (see, e.g., Jeger et al. (2010) Angew Chem. Int. Ed.Engl. 49: 9995-9997). By way of illustration, the glutamine tag, LLQG,has been engineered into different sites in the constant domain of anantibody targeting the epidermal growth factor receptor. mTG was thenused to conjugate these sites with fluorophores or monomethyl dolastatin10 (MMAD) and several sites where found to have good biophysicalproperties and a high degree of conjugation. mTG was also able toconjugate to glutamine tags on anti-Her2 and anti-M1S1 antibodies. AnantiM1S1-vc-MMAD conjugate displayed strong in vitro and in vivoactivity, suggesting that conjugation using this method does not alterantibody binding or affinity and demonstrates the utility of thisapproach in the site-specific conjugation of ADCs (see, e.g., Strop etal. (2013) Chem. Biol. 20: 161-167).

In addition to glycotransferases and transglutaminases, other enzymeshave been explored for use in protein labeling (Sunbul and Yin (2009)Org. Biomol. Chem. 7: 3361-3371). One such enzyme, formylglycinegenerating enzyme, recognizes the sequence CxPxR and oxidizes a cysteineresidue to form formylglycine, thus generating a protein with analdehyde tag. The aldehyde group can then be conjugated to molecule ofchoice through, e.g., hydrozino-Pictet-Spengler chemistry.

Many other procedures and linker molecules for attachment of variouscompounds including radionuclide metal chelates, toxins and drugs toproteins such as antibodies are known (see, e.g., European PatentApplication No. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148,4,699,784; 4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al.(1987) Cancer Res. 47: 4071-4075). In particular, production of variousimmunotoxins is well-known within the art and can be found, for examplein “Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet,”Thorpe et al., Monoclonal Antibodies in Clinical Medicine, AcademicPress, pp. 168-190 (1982), Waldmann (1991) Science, 252: 1657, U.S. Pat.Nos. 4,545,985 and 4,894,443.

In some circumstances, it is desirable to free the effector from theantibody when the immunoconjugate has reached its target site.Therefore, immunoconjugates comprising linkages that are cleavable inthe vicinity of the target site may be used when the effector is to bereleased at the target site. Cleaving of the linkage to release theagent from the antibody may be prompted by enzymatic activity orconditions to which the immunoconjugate is subjected either inside thetarget cell or in the vicinity of the target site. When the target siteis a tumor, a linker which is cleavable under conditions present at thetumor site (e.g. when exposed to tumor-associated enzymes or acidic pH)may be used.

A number of different cleavable linkers are known to those of skill inthe art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014.Illustrative cleavable linkers include, but are not limited to,acid-labile linkers, protease cleavable linkers, disulfide linkers, andthe like. Acid-labile linkers are designed to be stable at pH levelsencountered in the blood, but become unstable and degrade when the lowpH environment in lysosomes is encountered. Protease-cleavable linkersare also designed to be stable in blood/plasma, but rapidly release freedrug inside lysosomes in cancer cells upon cleavage by lysosomalenzymes. They take advantage of the high levels of protease activityinside lysosomes and typically include a peptide sequence that isrecognized and cleaved by these proteases, e.g., as occurs with adipeptide Val-Cit linkage that is rapidly hydrolyzed by cathepsins.Disulfide linkers exploit the high level of intracellular reducedglutathione to release free drug inside the cell.

In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, drugs, toxins, and other agents to antibodies one skilled inthe art will be able to determine a suitable method for attaching agiven agent to an antibody or other polypeptide.

Conjugation of Chelates.

In certain embodiments, the effector comprises a chelate that isattached to an antibody or to an epitope tag. The anti-CD46 antibodybears a corresponding epitope tag or antibody so that simple contactingof the antibody to the chelate results in attachment of the antibodywith the effector. The combining step can be performed before the moietyis used (targeting strategy) or the target tissue can be bound to theantibody before the chelate is delivered. Methods of producing chelatessuitable for coupling to various targeting moieties are well known tothose of skill in the art (see, e.g., U.S. Pat. Nos. 6,190,923,6,187,285, 6,183,721, 6,177,562, 6,159,445, 6,153,775, 6,149,890,6,143,276, 6,143,274, 6,139,819, 6,132,764, 6,123,923, 6,123,921,6,120,768, 6,120,751, 6,117,412, 6,106,866, 6,096,290, 6,093,382,6,090,800, 6,090,408, 6,088,613, 6,077,499, 6,075,010, 6,071,494,6,071,490, 6,060,040, 6,056,939, 6,051,207, 6,048,979, 6,045,821,6,045,775, 6,030,840, 6,028,066, 6,022,966, 6,022,523, 6,022,522,6,017,522, 6,015,897, 6,010,682, 6,010,681, 6,004,533, and 6,001,329).

Production of Fusion Proteins.

Where the antibody and/or the effector is relatively short (e.g., lessthan about 50 amino acids) they can be synthesized using standardchemical peptide synthesis techniques. Where both molecules arerelatively short the chimeric molecule may be synthesized as a singlecontiguous polypeptide. Alternatively the targeting molecule and theeffector molecule may be synthesized separately and then fused bycondensation of the amino terminus of one molecule with the carboxylterminus of the other molecule thereby forming a peptide bond.Alternatively, the targeting and effector molecules can each becondensed with one end of a peptide spacer molecule thereby forming acontiguous fusion protein.

Solid phase synthesis in which the C-terminal amino acid of the sequenceis attached to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence is the preferred method forthe chemical synthesis of the polypeptides of this invention. Techniquesfor solid phase synthesis are described by Barany and Merrifield,Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963), andStewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill. (1984).

In certain embodiments, the chimeric fusion proteins of the presentinvention are synthesized using recombinant DNA methodology. Generallythis involves creating a DNA sequence that encodes the fusion protein,placing the DNA in an expression cassette under the control of aparticular promoter, expressing the protein in a host, isolating theexpressed protein and, if required, renaturing the protein.

DNA encoding the fusion proteins of this invention can be prepared byany suitable method, including, for example, cloning and restriction ofappropriate sequences, or direct chemical synthesis by methods such asthe phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol.68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981)Tetra. Lett., 22: 1859-1862; and the solid support method of U.S. Pat.No. 4,458,066.

Chemical synthesis produces a single stranded oligonucleotide. This canbe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences can be obtained by the ligation of shorter sequences.

Alternatively, in certain embodiments subsequences can be cloned and theappropriate subsequences cleaved using appropriate restriction enzymes.The fragments can then be ligated to produce the desired DNA sequence.

In certain embodiments DNA encoding fusion proteins of the presentinvention can be cloned using PCR cloning methods.

While the antibody and the effector are, in certain embodiments,essentially joined directly together, one of skill will appreciate thatthe molecules can be separated by a spacer, e.g., a peptide spacerconsisting of one or more amino acids (e.g., (Gly₄Ser)₃, SEQ ID NO:80).Generally the spacer will have no specific biological activity otherthan to join the proteins or to preserve some minimum distance or otherspatial relationship between them. However, the constituent amino acidsof the spacer may be selected to influence some property of the moleculesuch as the folding, net charge, or hydrophobicity.

The nucleic acid sequences encoding the fusion proteins can be expressedin a variety of host cells, including E. coli, other bacterial hosts,yeast, and various higher eukaryotic cells such as the COS, CHO and HeLacells lines and myeloma cell lines. The recombinant protein gene will beoperably linked to appropriate expression control sequences for eachhost.

The plasmids of the invention can be transferred into the chosen hostcell by well-known methods such as calcium chloride transformation forE. coli and calcium phosphate treatment or electroporation for mammaliancells. Cells transformed by the plasmids can be selected by resistanceto antibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo and hyg genes.

Once expressed, the recombinant fusion proteins can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes (1982) ProteinPurification, Springer-Verlag, N.Y.; Deutscher (1990) Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y.). Substantially pure compositions of at least about 90 to 95%homogeneity are preferred, and 98 to 99% or more homogeneity are mostpreferred for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the polypeptides may then be usedtherapeutically.

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the fusion protein may possess aconformation substantially different than the native conformations ofthe constituent polypeptides. In this case, it may be necessary todenature and reduce the polypeptide and then to cause the polypeptide tore-fold into the preferred conformation. Methods of reducing anddenaturing proteins and inducing re-folding are well known to those ofskill in the art (see, e.g., Debinski et al. (1993) J. Biol. Chem., 268:14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4: 581-585;and Buchner, et al. (1992) Anal. Biochem., 205: 263-270).

One of skill would recognize that modifications can be made to thefusion proteins without diminishing their biological activity. Somemodifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids placed on either terminus tocreate conveniently located restriction sites or termination codons.

Pharmaceutical Compositions.

The anti-CD46 antibodies described herein (e.g., YS5, YS5F, YS5v1D,SB1HGNY, YS12, 3G7RY (aka 3G8), YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10,YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and/or UA8kappa) and/orimmunoconjugates thereof are useful for parenteral, topical, oral, orlocal administration (e.g. injected into a tumor site), aerosoladministration, or transdermal administration, for prophylactic, butprincipally for therapeutic treatment. The pharmaceutical compositionscan be administered in a variety of unit dosage forms depending upon themethod of administration. For example, unit dosage forms suitable fororal administration include powder, tablets, pills, capsules andlozenges. It is recognized that the antibodies described herein and/orimmunoconjugates thereof and pharmaceutical compositions comprisingantibodies described herein and/or immunoconjugates thereof, whenadministered orally, are preferably protected from digestion. This canbe accomplished by a number of means known to those of skill in the art,e.g., by complexing the protein with a composition to render itresistant to acidic and enzymatic hydrolysis or by packaging the proteinin an appropriately resistant carrier such as a liposome. Means ofprotecting proteins from digestion are well known in the art.

In various embodiments a composition, e.g., a pharmaceuticalcomposition, containing one or a combination of anti-CD46 antibodies, orantigen-binding portion(s) thereof, or immunoconjugates thereof,formulated together with a pharmaceutically acceptable carrier areprovided.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjugate, may be coated in a material to protect the compoundfrom the action of acids and other natural conditions that mayinactivate the compound.

In certain embodiments the antibody and/or immunoconjugate can beadministered in the “native” form or, if desired, in the form of salts,esters, amides, prodrugs, derivatives, and the like, provided the salt,ester, amide, prodrug or derivative is suitable pharmacologically, i.e.,effective in the present method(s). Salts, esters, amides, prodrugs andother derivatives of the active agents can be prepared using standardprocedures known to those skilled in the art of synthetic organicchemistry and described, for example, by March (1992) Advanced OrganicChemistry; Reactions, Mechanisms and Structure, 4th Ed. N.Y.Wiley-Interscience, and as described above.

By way of illustration, a pharmaceutically acceptable salt can beprepared for any of the antibodies and/or immunoconjugates describedherein having a functionality capable of forming a salt. Apharmaceutically acceptable salt is any salt that retains the activityof the parent compound and does not impart any deleterious or untowardeffect on the subject to which it is administered and in the context inwhich it is administered.

In various embodiments pharmaceutically acceptable salts may be derivedfrom organic or inorganic bases. The salt may be a mono or polyvalention. Of particular interest are the inorganic ions, lithium, sodium,potassium, calcium, and magnesium. Organic salts may be made withamines, particularly ammonium salts such as mono-, di- and trialkylamines or ethanol amines. Salts may also be formed with caffeine,tromethamine and similar molecules.

Methods of formulating pharmaceutically active agents as salts, esters,amide, prodrugs, and the like are well known to those of skill in theart. For example, salts can be prepared from the free base usingconventional methodology that typically involves reaction with asuitable acid. Generally, the base form of the drug is dissolved in apolar organic solvent such as methanol or ethanol and the acid is addedthereto. The resulting salt either precipitates or can be brought out ofsolution by addition of a less polar solvent. Suitable acids forpreparing acid addition salts include, but are not limited to bothorganic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvicacid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like, as well asinorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, and the like. An acid addition saltcan be reconverted to the free base by treatment with a suitable base.Certain particularly preferred acid addition salts of the active agentsherein include halide salts, such as may be prepared using hydrochloricor hydrobromic acids. Conversely, preparation of basic salts of theactive agents of this invention are prepared in a similar manner using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or thelike. Particularly preferred basic salts include alkali metal salts,e.g., the sodium salt, and copper salts.

For the preparation of salt forms of basic drugs, the pKa of thecounterion is preferably at least about 2 pH units lower than the pKa ofthe drug. Similarly, for the preparation of salt forms of acidic drugs,the pKa of the counterion is preferably at least about 2 pH units higherthan the pKa of the drug. This permits the counterion to bring thesolution's pH to a level lower than the pH_(max) to reach the saltplateau, at which the solubility of salt prevails over the solubility offree acid or base. The generalized rule of difference in pKa units ofthe ionizable group in the active pharmaceutical ingredient (API) and inthe acid or base is meant to make the proton transfer energeticallyfavorable. When the pKa of the API and counterion are not significantlydifferent, a solid complex may form but may rapidly disproportionate(i.e., break down into the individual entities of drug and counterion)in an aqueous environment.

Preferably, the counterion is a pharmaceutically acceptable counterion.Suitable anionic salt forms include, but are not limited to acetate,benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate,edetate, edisylate, estolate, fumarate, gluceptate, gluconate,hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate,maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate,napsylate, nitrate, pamoate (embonate), phosphate and diphosphate,salicylate and disalicylate, stearate, succinate, sulfate, tartrate,tosylate, triethiodide, valerate, and the like, while suitable cationicsalt forms include, but are not limited to aluminum, benzathine,calcium, ethylene diamine, lysine, magnesium, meglumine, potassium,procaine, sodium, tromethamine, zinc, and the like.

Preparation of esters typically involves functionalization of hydroxyland/or carboxyl groups that are present within the molecular structureof the antibody and/or immunoconjugate. In certain embodiments, theesters are typically acyl-substituted derivatives of free alcoholgroups, i.e., moieties that are derived from carboxylic acids of theformula RCOOH where R is alky, and preferably is lower alkyl. Esters canbe reconverted to the free acids, if desired, by using conventionalhydrogenolysis or hydrolysis procedures.

Amides can also be prepared using techniques known to those skilled inthe art or described in the pertinent literature. For example, amidesmay be prepared from esters, using suitable amine reactants, or they maybe prepared from an anhydride or an acid chloride by reaction withammonia or a lower alkyl amine.

Pharmaceutical compositions comprising the antibodies and/orimmunoconjugates described herein can be administered alone or incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include a an antibody or immunoconjugate with atleast one or more additional therapeutic agents, such as the anti-canceragents described infra. The pharmaceutical compositions can also beadministered in conjunction with radiation therapy and/or surgery.

A composition comprising the antibodies and/or immunoconjugatesdescribed herein can be administered by a variety of methods known inthe art. As will be appreciated by the skilled artisan, the route and/ormode of administration will vary depending upon the desired results. Theactive compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art (see, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978).

In certain embodiments administration of an anti-CD46 antibody orimmunoconjugate may be facilitated by coating the antibody orimmunoconjugate composition, or co-administering the antibody orimmunoconjugate, a material to prevent its inactivation. For example,the compound may be administered to a subject in an appropriate carrier,for example, liposomes, or a diluent. Pharmaceutically acceptablediluents include, but are not limited to, saline and aqueous buffersolutions. Liposomes include, but are not limited to,water-in-oil-in-water CGF emulsions as well as conventional liposomes(Strejan et al. (1984) J. Neuroimmunol, 7: 27).

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

In various embodiments the therapeutic compositions are typicallysterile and stable under the conditions of manufacture and storage. Thecomposition(s) can be formulated as a solution, a microemulsion, in alipid or liposome, or other ordered structure suitable to contain highdrug concentration(s). In certain embodiments the carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., antibodies and/or immunoconjugates described herein) inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, illustrative methods of preparationinclude vacuum drying, and freeze-drying (lyophilization) that yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. For example, in certainembodiments, the antibodies and/or immunoconjugates described herein maybe administered once or twice daily, or once or twice weekly, or once ortwice monthly by subcutaneous injection.

It is especially advantageous to formulate parenteral compositions inunit dosage form for ease of administration and uniformity of dosage.Unit dosage form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated. Each unitcontains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specifications for the unit dosage forms aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of individuals.

In certain embodiments the formulation comprises a pharmaceuticallyanti-oxidant. Examples of pharmaceutically-acceptable antioxidantsinclude: (1) water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the antibodies and/orimmunoconjugates described herein include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods knownin the art of pharmacy. The amount of active ingredient which can becombined with a carrier material to produce a single dosage form willvary depending upon the subject being treated, and the particular modeof administration. The amount of active ingredient that can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the composition which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about0.001 percent to about ninety percent of active ingredient, preferablyfrom about 0.005 percent to about 70 percent, most preferably from about0.01 percent to about 30 percent.

Formulations of antibodies and/or immunoconjugates described herein thatare suitable for vaginal administration also include pessaries, tampons,creams, gels, pastes, foams or spray formulations containing suchcarriers as are known in the art to be appropriate. Dosage forms for thetopical or transdermal administration of antibodies and/orimmunoconjugates described herein include powders, sprays, ointments,pastes, creams, lotions, gels, solutions, patches and inhalants. Incertain embodiments the active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants that may be required.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection, andinfusion.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions comprising antibodies and/orimmunoconjugates described herein include, but are not limited to water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), and suitable mixtures thereof, vegetable oils,such as olive oil, and injectable organic esters, such as ethyl oleate,and the like. Proper fluidity can be maintained, for example, by the useof coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

In various embodiments these compositions may also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents and dispersingagents. Particular examples of adjuvants that are well-known in the artinclude, for example, inorganic adjuvants (such as aluminum salts, e.g.,aluminum phosphate and aluminum hydroxide), organic adjuvants (e.g.,squalene), oil-based adjuvants, virosomes (e.g., virosomes that containa membrane-bound hemagglutinin and neuraminidase derived from theinfluenza virus).

Prevention of presence of microorganisms in formulations may be ensuredboth by sterilization procedures, and/or by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

When the antibodies and/or immunoconjugates described herein areadministered as pharmaceuticals, to humans and animals, they can begiven alone or as a pharmaceutical composition containing, for example,0.001 to 90% (more preferably, 0.005 to 70%, such as 0.01 to 30%) ofactive ingredient in combination with a pharmaceutically acceptablecarrier.

Regardless of the route of administration selected, the antibodiesand/or immunoconjugates described herein, that may be used in a suitablehydrated form, and/or the pharmaceutical compositions, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients (e.g., antibodies and/orimmunoconjugates described herein) in the pharmaceutical compositions ofthe present invention may be varied so as to obtain an amount of theactive ingredient which is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts. A physician orveterinarian having ordinary skill in the art can readily determine andprescribe the effective amount of the pharmaceutical compositionrequired. For example, the physician or veterinarian could start dosesof the compounds of the invention employed in the pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. In general, a suitable daily dose ofantibodies and/or immunoconjugates described herein will be that amountof the compound which is the lowest dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above. In certain embodiments, it is preferredthat administration be intravenous, intramuscular, intraperitoneal, orsubcutaneous, preferably administered proximal to the site of thetarget. If desired, the effective daily dose of a therapeuticcomposition may be administered a single dosage, or as two, three, four,five, six or more sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms. While itis possible for antibodies and/or immunoconjugates described herein tobe administered alone, it is typically preferable to administer thecompound(s) as a pharmaceutical formulation (composition).

In certain embodiments the therapeutic compositions can be administeredwith medical devices known in the art. For example, in a illustrativeembodiment, antibodies and/or immunoconjugates described herein can beadministered with a needleless hypodermic injection device, such as thedevices disclosed in U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335,5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of usefulwell-known implants and modules are described for example in U.S. Pat.No. 4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate, in U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicationsthrough the skin, in U.S. Pat. No. 4,447,233, which discloses amedication infusion pump for delivering medication at a precise infusionrate, in U.S. Pat. No. 4,447,224, which discloses a variable flowimplantable infusion apparatus for continuous drug delivery, in U.S.Pat. No. 4,439,196, which discloses an osmotic drug delivery systemhaving multi-chamber compartments, and in U.S. Pat. No. 4,475,196, whichdiscloses an osmotic drug delivery system. Many other such implants,delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the anti-CD46 antibodies and/or immunoconjugatesdescribed herein can be formulated to ensure proper distribution invivo. For example, the blood-brain barrier (BBB) excludes many highlyhydrophilic compounds. To ensure that the therapeutic compounds of theinvention cross the BBB (if desired), they can be formulated, forexample, in liposomes. For methods of manufacturing liposomes, see,e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomesmay comprise one or more moieties which are selectively transported intospecific cells or organs, thus enhance targeted drug delivery (see,e.g., Ranade (1989) J. Clin. Pharmacol. 29: 685). Illustrative targetingmoieties include, but are not limited to folate or biotin (see, e.g.,U.S. Pat. No. 5,416,016); mannosides (Umezawa et al., (1988) Biochem.Biophys. Res. Commun. 153: 1038); antibodies (Bloeman et al. (1995) FEBSLett. 357:140; Owais et al. (1995) Antimicrob. Agents Chemother.39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J.Physiol. 1233:134).

Kits.

Where a radioactive, or other, effector is used as a diagnostic and/ortherapeutic agent, it is frequently impossible to put the ready-for-usecomposition at the disposal of the user, because of the often poor shelflife of the radiolabeled compound and/or the short half-life of theradionuclide used. In such cases the user can carry out the labelingreaction with the radionuclide in the clinical hospital, physician'soffice, or laboratory. For this purpose, or other purposes, the variousreaction ingredients can then be offered to the user in the form of aso-called “kit”. The kit is preferably designed so that themanipulations necessary to perform the desired reaction should be assimple as possible to enable the user to prepare from the kit thedesired composition by using the facilities that are at his disposal.Therefore the invention also relates to a kit for preparing acomposition according to this invention.

In certain embodiments, such a kit comprises one or more antibodies orimmunoconjugates described herein. The antibodies or immunoconjugatescan be provided, if desired, with inert pharmaceutically acceptablecarrier and/or formulating agents and/or adjuvants is/are added. Inaddition, the kit optionally includes a solution of a salt or chelate ofa suitable radionuclide (or other active agent), and (iii) instructionsfor use with a prescription for administering and/or reacting theingredients present in the kit.

The kit to be supplied to the user may also comprise the ingredient(s)defined above, together with instructions for use, whereas the solutionof a salt or chelate of the radionuclide, defined sub (ii) above, whichsolution has a limited shelf life, may be put to the disposal of theuser separately.

The kit can optionally, additionally comprise a reducing agent and/or,if desired, a chelator, and/or instructions for use of the compositionand/or a prescription for reacting the ingredients of the kit to formthe desired product(s). If desired, the ingredients of the kit may becombined, provided they are compatible.

In certain embodiments, the immunoconjugate can simply be produced bycombining the components in a neutral medium and causing them to react.For that purpose the effector may be presented to the antibody, forexample, in the form of a chelate.

When kit constituent(s) are used as component(s) for pharmaceuticaladministration (e.g. as an injection liquid) they are preferablysterile. When the constituent(s) are provided in a dry state, the usershould preferably use a sterile physiological saline solution as asolvent. If desired, the constituent(s) may be stabilized in theconventional manner with suitable stabilizers, for example, ascorbicacid, gentisic acid or salts of these acids, or they may comprise otherauxiliary agents, for example, fillers, such as glucose, lactose,mannitol, and the like.

While the instructional materials, when present, typically comprisewritten or printed materials they are not limited to such. Any mediumcapable of storing such instructions and communicating them to an enduser is contemplated by this invention. Such media include, but are notlimited to electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical media (e.g., CD ROM), and the like. Suchmedia may include addresses to internet sites that provide suchinstructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Novel Anti-CD46 Antibodies and Uses Thereof

To identify novel anti-human CD46 antibodies, a recombinant Fc fusionprotein composed of the Sushi domain 1 and 2 of human CD46 was created.As complement elements bind predominantly to domain 3 and 4, the choiceof domain 1 and 2 minimize selection of antibodies that could potentlyinterfere with normal complement function. This CD46-Fc fusion wasproduced and purified from transfected HEK293 cells by protein Aaffinity chromatography. For human antibody selection, a 5×10⁹ memberphagemid display library was created using cDNAs pooled from peripheralblood mononuclear cells of 426 healthy human donors, and the library wasselected the recombinant CD46-Fc fusion protein. Following three roundsof selection, binding phagemid were screened by FACS and sequenced. Inparallel, an alternatively strategy was employed that involve firstselecting the library on live tumor cells followed by transferring theoutput from round 1 phagemid selection into a yeast surface displayvector, and then FACS-based selection using low concentration ligands toenrich high affinity binders to the recombinant CD46-Fc fusion protein.The resulting antibodies bind with high affinity to both live tumorcells and the recombinant human CD46 protein. All binding clones weresequenced and unique sequences are listed in Table 1.

The scFvs were converted into full human IgG1s and binding affinitieswere measured on live tumor cells. FACS binding data were curved fit togenerate KD values (FIG. 2 for YS5 on Du-145, and FIG. 3 for YS12 onDu-145). Affinities range from low to sub-nanomolar (nM) for theantibodies studied.

In addition to human CD46, the antibodies described herein bind to thecynomolgus monkey CD46 (FIG. 4) with similar affinities (FIG. 5 for YS5on CHO-huCD46 and FIG. 6 YS5 on CHO-cynoCD46), thus identifying anappropriate species for regulatory toxicology studies.

All of the anti-CD46 antibodies, along with the previously identifiedUA20 and 2B10, bind to CD46 Sushi domains 1 and 2. This region wasfurther analyzed to reveal epitope differences. We first performedFACS-based competition experiments on live tumor cells and found thatour antibodies either compete or do not compete with UA20-Fc: Group 1consists of YS5 and others, and Group 1 consists of SB1HGNY (FIG. 7).For antibodies in Group 1, there are additional epitope differences asevidenced by selective effect of antibody binding to various CD46mutants (Table 3). For example, YS5 differs from other antibodies asmutation in position 39 uniquely affects its binding (Table 3),suggesting that the position 39, along with the position 40 that impactsbinding for all antibodies, is part of the epitope for YS5 binding toCD46.

TABLE 3 Alanine scan reveals epitope differences. CD46 residue changesare as indicated (top row, E13A, position 13 changed from E to A, etc).Binding to CHO cells transfected with various mutant constructed werequantified by FACS with MFI normalized against wild typeCD46-transfected cells. A significant loss of binding is shown. R40Areduced binding for all antibodies, indicating that position 40 is acritical contact site for all. D39A uniquely affects YS5 binding,indicating the position 39 is a unique contact site that contributes toYS5 (and YS6) binding, but not other antibodies listed in the table. YS6differs from YS5 as it is selectively affected by position 31 mutation(P31A). E13A E16A P31A T36A D39A R40A YS5 120 124 111 104 76 56 YS12 96157 89 88 121 77 UA20FC 133 123 113 136 120 73 3G8 212 132 117 83 129 70YS6 90 96 57 111 81 65 2B10 81 113 86 104 107 76

All the antibodies compete with the laboratory strain (Edmonston)measles virus H protein. Measles virus enters target cells bymacropinocytosis (Crimeen-Irwin et al. (2003) J. Biol. Chem. 278:46927-46937). The H protein is responsive for binding and crosslinkingcell surface CD46, which is required for viral entry (Id.). To determineif our antibodies compete with H protein binding to CD46, a recombinantH protein-Fc fusion was created, and tested by FACS for competition withthe anti-CD46 antibodies. It was found that the antibodies testedcompete with H protein (FIG. 8), suggesting overlapping binding sites.

As measles virus enters target cells through macropinocytosis, it wasnext determined if the anti-CD46 antibodies are also internalized bytumor cells via maicropinocytosis. Using a 70 kDa neutral densitydextran (ND70) as an indicator for macropinocytosis (Ha et al. (2014)Mol. Cell Proteomics. 13(12): 3320-3331), it was found that theanti-CD46 antibodies are indeed internalized by the macropinocytosispathway (FIG. 9). Macropinocytosis is inherently tumor selective(Commisso et al. (2013) Nature, 497: 633-637; Ha et al. (2014) Mol. CellProteomics. 13(12): 3320-3331; Reyes-Reyes et al. (2010) Cancer Res. 70:8617-8629), thus imparting the anti-CD46 antibodies with an additionalselectivity for tumor cells.

To validate CD46 as a target for targeted therapeutic development,tissue specificity of the CD46 epitope was determined byimmunohistochemistry. CD46 epitope expression in tumor was firststudied. Immunohistochemistry studies were performed on both frozen andformalin fixed paraffin embedded (FFPE) prostate cancer tissues. Onfrozen tissues, 18/18 cases (100%) showed strong staining signals,indicating overexpression in all cases. On FFPE tissues, strong CD46staining was found in 63/87, moderate staining in 23/87, and weakstaining in 1/87 cases, supporting the conclusion that CD46 isoverexpressed by a vast majority of prostate tumors. A summary of theimmunohistochemistry results is shown in Table 4 and FIG. 10.

TABLE 4 Immunohistochemistry results of CD46 expression in prostatecancer tissues. Positive vs. total cases studied are indicated. FFPE:formalin fixed paraffin embedded (tissues). Biotin-labeled anti-humanCD46 antibodies were used for this study. Tissue type Strong ModerateWeak Negative Frozen 18/18 0 0 0 FFPE 63/87 23/87 1/87 0

The aforementioned studies strongly support the development of aCD46-targeted monoclonal antibody therapeutic against human cancer. Tothis end, antibody drug conjugates (ADCs) were developed using the panelof novel anti-CD46 antibodies. Monomethyl auristatin F (MMAF) wasconjugated via the MC-vc-PAB linker (McDonagh et al. (2008) Mol. Canc.Therap. 7: 2913-2923; Sutherland et al. (2006) J. Biol. Chem. 281:10540-10547) to YS5 IgG1. By hydrophobic interaction chromatography(HIC), it was determined that, on average, about 3 drugs were conjugatedper antibody molecule (FIG. 11). Tumor-killing activities in vitro werethen tested using a panel of metastatic castration resistant prostatecancer cell lines (LNCaP-C4-2B and Du145). Potent tumor cell killing wasobserved with EC50 values ranging from low to sub nM (FIGS. 12 and 13).

In vivo anti-tumor activity of the anti-CD46 ADCs was tested using theLNCaP-C4-2B subcutaneous xenograft model. Potent inhibition of tumorgrowth and survival was observed, with tumor volume reduced tonon-detectable levels following 5 doses at 5 mg/kg (FIG. 14). No tumorrecurred during the indicated period post ADC injection (FIG. 14).

In addition to prostate cancer, it was found that the anti-CD46 ADCspotently kill various other cancers that express CD46. For example, itwas found that in addition to prostate cancer, CD46 is also highlyexpressed on multiple myeloma cell surface (FIG. 15). Moreover, theanti-CD46 ADC potently kills the multiple myeloma cell line RPMI8226(FIG. 16) with EC50 in the sub nM range. Finally it was shown that theanti-CD46 ADC greatly reduced multiple myeloma burdens in vivo. Areporter-expressing line (RPMI8226-Luc that expressed the fireflyluciferase gene) was injected via the tail vein to immune-compromisedmice and the disseminated tumor cells were allowed to establish in thebone and joints. Four doses of anti-CD46 ADCs were then injected at 5mg/kg (every 4 days) and tumor status was monitored by bioluminescence.It was found that the anti-CD46 ADCs are highly effective in reducingtumor burdens in vivo (FIG. 17). Survival data were collected followingADC treatment (FIG. 18), showing “cure” for 60% of the treated mice andgreatly delayed onset of death for the remaining 40% through theduration of the experiment.

To broaden applicability, the anti-CD46 ADC was studied using a secondmultiple myeloma cell line MM1.S that expresses the luciferase reporter.Potent tumor killing activity of the anti-CD46 ADC was observed in vivo.Four doses at 4 mg/kg completely eliminated MM1.S tumor cells (FIG. 20).In contrast, the control ADC (MMAF conjugated to a non-binding antibody)had no effect at all at the same dose and dosing frequency. Moreover,even a single dose of anti-CD46 ADC at 4 mg/kg greatly reduced tumorburden, causing a profound inhibition of tumor development (FIG. 20).Four doses of anti-CD46 ADC at 0.8 mg/kg also caused a lastinginhibition of MM1.S xenograft development (FIG. 20). Finally, even nakedantibody (YS5 IgG1) caused significant tumor inhibition in thisxenograft model (FIG. 20), suggesting an interesting possibility of apotential naked antibody-based therapy for certain subtypes of multiplemyeloma.

Kaplan-Meier analysis was performed to determine survivalpost-treatment. CD46 ADC treated groups showed a significant survivaladvantage over control groups (FIG. 21). For mice that received 4 dosesof 4 mg/kg CD46 ADC, all survived until the end of the experiment (day212).

Besides the aforementioned prostate cancer and multiple myeloma, it wasfound that CD46 is highly expressed in a broad panel of cancer celllines including, but not limited to, colorectal cancer, pancreaticcancer, mesothelioma, lung cancer, breast cancer, ovarian cancer,bladder cancer, liver cancer, glioma, and neuroblastoma. Moreover, theanti-CD46 ADCs potently killed those cells in vitro. For example, theanti-CD46 ADC is highly effective in killing colorectal cancer cells(FIG. 19 for YS5, FIG. 22 for YS12 on HT29, and FIG. 23 for SB1HGNY onHT29), pancreatic cancer cells (FIG. 24), mesothelioma cells (FIG. 25),and ovarian cancer cells (FIG. 26).

To evaluate potential toxicity, YS5 ADC was tested on a panel of controlcells that either do not express CD46 (BPH-1) or that express it atmoderate levels (e.g., HS27). Much reduced cytotoxicity was observed onthose cells (FIG. 27 for YS5 ADC on BPH-1, FIG. 28 for YS5 ADC on HS27,FIG. 29 for YS5 ADC on normal T cells, and FIG. 30 for YS5 ADC onCD14-depleted peripheral blood mononuclear cells (PBMC)), suggestingthat they do not take up readily the ADCs. Differential internalizationvia macropinocytosis is a likely mechanism that enhances selectivity ofanti-CD46 ADCs.

ADC toxicity was studied in vivo using transgenic mice that expresshuman CD46. There is not a true murine ortholog for human CD46 andmurine CD46 performs a quite different physiological role than that ofhuman CD46. The antibodies did not bind to murine CD46. Thus there is nogood small animal model for evaluation of potential anti-CD46 ADCtoxicity except for the transgenic model. Anti-CD46 ADC was injected at6 mg/kg into human CD46-expressing transgenic mice, and the animals weremonitored for overt sign of toxicity daily. The animals were sacrificedat day 14 and the vital organs were harvested for histologicalexamination of tissue damages. As shown in FIG. 31, there is no notabledifference between the anti-CD46 ADC-treated and the control ADC-treatedmice. No overt sign of toxicity was observed during the duration of thisexperiment at this dose of ADC tested. It is understood that regulatorytoxicology studies need to be performed in non-human primates such asthe cynomolgus monkey whose CD46 is recognized by the anti-CD46antibodies as shown above.

Given that human CD46 gene is located at the short arm of chromosome 1(1q32.2), and given that 1q gain has been frequently observed in avariety of cancers especially those with poor prognosis, it is likelythat anti-CD46 antibody therapeutics including but not limited to ADCsare applicable to a broad spectrum of malignancies at advanced stageswith dire therapeutic need. For example, in prostate cancer, the regionspanning 1q32.2 has been shown to gain in disseminated metastatic formof the disease where current therapies have failed to make an impact,(Hanamura et al. (2006) Blood, 108: 1724-1732), making metastaticcastration resistant prostate cancer an excellent candidate for ouranti-CD46 ADC treatment. In multiple myeloma, 1q gain is also frequentlyobserved in recurrent patients (Id.), thus identifying an importantpatient population that our anti-CD46 ADCs could potentially help. Thecorrelation between 1q gain (and specifically 1q32.2) and poor prognosishas been seen for other cancers as well, making 1q a potential biomarkerfor our anti-CD46 ADCs for patient stratification and outcome monitoringfor those malignancies. FISH probe detecting 1q32.2 could be used toassess 1q status. Circulating DNA could also be used to detect 1q gainas a minimally invasive biomarker (Fan et al. (2008) Proc. Natl. Acad.Sci. USA, 105: 16266-16271) for CD46-expressing cancers with poorprognosis.

The anti-CD46 antibodies described herein can be used to in an imagingprobe to monitor tumor status in vivo, either as a standalone imagingagent or a companion diagnostic for the anti-CD46 ADCs. We havepreviously labeled our original anti-CD46 antibody UA20 and demonstratedits excellent imaging property in vivo for prostate cancer targeting (Heet al. (2010) J. Nucl. Med. 51: 427-432). Besides imaging, the anti-CD46antibodies can in immunohistochemistry-based biomarkers to assess CD46expression in biopsies and archived patient samples, capture ELISAassays to assess serum CD46 levels, and FACS or chip-based assays toassess CD46 cell surface expression in disseminated and/or circulatingtumor cells.

Example 2

CD46 ADC is Highly Active in Intra-Femoral mCRPC Xenograft Model.

As over 95% of prostate cancer metastasis is to the site of the bone, wefurther studied efficacy of our anti-CD46 ADC in a bone xenograft model.We injected the metastatic castration resistant prostate cancer (mCRPC)cell line LNCaP C4-2B that carries a firefly luciferase reporter intothe femur of NSG mice to create the intra-bone mCRPC xenograft model.The CD46 ADC (YS5-mcvcpab-MMAF) was injected 7 days post grafting every4 days for a total of 4 times. Tumor status was monitored bybioluminescence imaging during and post treatment. As shown in FIG. 32,CD46 ADC treated mice showed profound tumor inhibition that last throughthe post-treatment period til the end of the experiment (day 65),suggesting that our CD46 ADC is highly efficacious in this intra-femoralmCRPC xenograft model.

CD46 is Highly Expressed in CRPC and mCRPC Tissues.

In addition to primary tumors, we performed immunohistochemistry studieson tissue specimens from castration resistant prostate cancer (CRPC) andmetastatic castration resistant prostate cancer (mCRPC). As shown inFIG. 33, CD46 is highly expressed in CRPC specimens. We further studiedmCRPC specimens and found widespread (100% of cases studied, or 12/12)and strong expression of CD46 in bone metastasis (FIG. 34), lymph nodemetastasis (FIG. 35), and bladder metastasis (FIG. 36).

CD46 is Overexpressed by Neuroendocrine Subtype of Prostate Cancer.

About 30% patients are resistant to abiraterone and enzalutamidetreatment. Emergence of small cell/neuroendocrine type of prostatecancer may be a frequent event (˜30-40% of cases). Unlikeadenocarcinoma, neuroendocrine prostate cancer often do not expresscommon markers such as prostate specific antigen (PSA) and prostatespecific membrane antigen (PSMA). Therefore we sought to study by FACSCD46 expression by a neuroendocrine prostate cancer cell line H660. Asshown in FIG. 37, left panel, CD46 is highly expressed by H660 cells.Western blot analysis confirmed that H660 cells express CD46 and theneuroendocrine marker neuron-specific enolase (NSE) (FIG. 37, rightpanel). Our anti-CD46 antibody (YS5) is internalized by H660 cells andco-localized with the lysosomal marker LAMP1 (FIG. 38). When incubatedwith H660 cells, our anti-CD46 ADC showed potent cytotoxic activities invitro with EC50<1 nM (FIG. 39).

CD46 is Further Upregulated by Prostate Cancer Cells Following Treatmentwith Abiraterone or Enzalutamide, Rendering Them Susceptible to CD46ADC.

We found that treating the mCRPC line LNCaP-C4-2B with 10 μM abirateronefor 7 days caused a significant upregulation of surface CD46 expression(FIG. 40). Interestingly, this upregulation correlates with an enhancedkilling of tumor cells (FIG. 41) with EC50 values dropping from 169 pMto 21 pM. Similarly, when the neuroendocrine prostate cancer cell lineH660 was incubated with 10 μM enzalutamide for 7 days, a significantupregulation of cell surface CD46 was observed (FIG. 42). Like what wasobserved in LNCaP-C4-2B cells, H660 cells became more sensitive to CD46ADC post enzalutamide treatment with EC50 dropping by 4-5 fold.

CD46 Expression on Additional Tumors.

In addition to prostate cancer and multiple myeloma, we found that CD46is overexpressed in a wide range of human cancers. Byimmunohistochemistry analysis, we found positive CD46 staining in 82% ofcolorectal cancer (81/99 cases) with 70/99 showing strong staining (71%)(FIG. 43). Interestingly, nearly 100% of metastatic colorectal cancersexpress CD46 (liver metastasis in FIG. 44, lymph node metastasis in FIG.45, and bladder metastasis in FIG. 46).

Positive CD46 staining was also observed for 41/50 cases (82%) ofmesothelioma with 31/50 showing strong staining (62%) (FIG. 47). Inpancreatic cancer, positive CD46 staining was observed in 28/50 cases(56%) (FIG. 48). In glioblastoma multiforme (GBM), positive staining wasobserved in 30/40 (75%) cases (FIG. 49).

Positive staining was also observed in other tumors including but arenot limited to bladder cancer, ovarian cancer, stomach cancer, lungcancer, liver cancer, breast cancer, and lymphoma.

CD46 ADC is Effective Against Other Tumors.

In addition to in vitro studies, we performed an in vivo study of CD46ADC on mesothelioma xenografts carried in NSG mice. As shown in FIG. 50,YS5-mcvcpab-MMAF is highly effective in inhibiting tumor xenograftdevelopment.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. An isolated recombinant human antibody thatspecifically binds CD46 and is internalized, via macropinocytosis, intoa cell expressing or overexpressing CD46, wherein: said isolatedrecombinant human antibody specifically binds cells that express oroverexpress a CD46, and wherein said isolated recombinant human antibodycomprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of anantibody selected from the group consisting of YS5 (represented by SEQID NOs: 1 and 22), YS5F (represented by SEQ ID NOs: 2 and 23), YS5v1D(represented by SEQ ID NOs: 3 and 24), SB1HGNY (represented by SEQ IDNOs: 4 and 25), YS12 (represented by SEQ ID NOs: 5 and 26), 3G7RY(represented by SEQ ID NOs: 6 and 27), YS6 (represented by SEQ ID NOs: 7and 28), YS1 (represented by SEQ ID NOs: 8 and 29), YS3 (represented bySEQ ID NOs: 9 and 30), YS4 (represented by SEQ ID NOs: 10 and 31), YS8(represented by SEQ ID NOs: 11 and 32), YS7 (represented by SEQ ID NOs:12 and 33), YS9 (represented by SEQ ID NOs: 13 and 34), YS10(represented by SEQ ID NOs: 14 and 35), YS11 (represented by SEQ ID NOs:15 and 36), 3G7HY (represented by SEQ ID NOs: 16 and 37), 3G7NY(represented by SEQ ID NOs: 17 and 38), 3G7 (represented by SEQ ID NOs:18 and 39), SB2 (represented by SEQ ID NOs: 19 and 40), 2C8 (representedby SEQ ID NOs: 20 and 41), or UA8kappa (represented by SEQ ID NOs: 21and 42).
 2. The antibody of claim 1, wherein said cells that express oroverexpress a CD46 are cancer cells.
 3. The antibody of claim 1, whereinsaid cells that express or overexpress a CD46 are prostate cancer cells.4. The antibody of claim 1, wherein said isolated recombinant humanantibody is an intact immunoglobulin.
 5. The antibody of claim 4,wherein said isolated recombinant human antibody comprises an IgA, IgE,or IgG.
 6. The antibody of claim 4, wherein said isolated recombinanthuman antibody comprises an IgG1.
 7. The antibody of claim 1, whereinsaid isolated recombinant human antibody is an antibody fragment thatspecifically binds cells that express or overexpress a CD46.
 8. Theantibody of claim 7, wherein said isolated recombinant human antibody isan antibody fragment selected from the group consisting of Fv, Fab,(Fab′)₂, (Fab′)₃, IgGΔCH2, and a minibody.
 9. The antibody of claim 1,wherein said isolated recombinant human antibody is a single chainantibody.
 10. The antibody of claim 1, wherein said isolated recombinanthuman antibody comprises the variable light (VL) chain of an antibodyselected from the group consisting of YS5, YS5F, YS5v1D, SB1HGNY, YS12,3G7RY, YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10, YS11, 3G7HY, 3G7NY, 3G7,SB2, 2C8, and UA8kappa.
 11. The antibody of claim 1, wherein saidisolated recombinant human antibody comprises the variable heavy (VH)chain of an antibody selected from the group consisting of YS5, YS5F,YS5v1D, SB1HGNY, YS12, 3G7RY, YS6, YS1, YS3, YS4, YS8, YS7, YS9, YS10,YS11, 3G7HY, 3G7NY, 3G7, SB2, 2C8, and UA8kappa.
 12. The antibody ofclaim 1, wherein said isolated recombinant human antibody comprises VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the YS5antibody.
 13. The antibody of claim 1, wherein said isolated recombinanthuman antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2,and VL CDR3 of the YS5F antibody.
 14. The antibody of claim 1, whereinsaid isolated recombinant human antibody comprises VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2, and VL CDR3 of the YS5v1D antibody.
 15. Theantibody of claim 1, wherein said isolated recombinant human antibodycomprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 ofthe SB1HGNY antibody.
 16. The antibody of claim 1, wherein said isolatedrecombinant human antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VL CDR2, and VL CDR3 of the YS12 antibody.
 17. The antibody of claim 1,wherein said isolated recombinant human antibody comprises VH CDR1, VHCDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the 3G7RY antibody. 18.The antibody of claim 1, wherein said isolated recombinant humanantibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3 of the YS6 antibody.
 19. The antibody of claim 1, wherein saidisolated recombinant human antibody comprises VH CDR1, VH CDR2, VH CDR3,VL CDR1, VL CDR2, and VL CDR3 of the YS1 antibody.
 20. The antibody ofclaim 1, wherein said isolated recombinant human antibody comprises VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the YS3antibody.
 21. The antibody of claim 1, wherein said isolated recombinanthuman antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2,and VL CDR3 of the YS4 antibody.
 22. The antibody of claim 1, whereinsaid isolated recombinant human antibody comprises VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2, and VL CDR3 of the YS8 antibody.
 23. Theantibody of claim 1, wherein said isolated recombinant human antibodycomprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 ofthe YS7 antibody.
 24. The antibody of claim 1, wherein said isolatedrecombinant human antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VL CDR2, and VL CDR3 of the YS9 antibody.
 25. The antibody of claim 1,wherein said isolated recombinant human antibody comprises VH CDR1, VHCDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the YS10 antibody. 26.The antibody of claim 1, wherein said isolated recombinant humanantibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3 of the YS11 antibody.
 27. The antibody of claim 1, wherein saidisolated recombinant human antibody comprises VH CDR1, VH CDR2, VH CDR3,VL CDR1, VL CDR2, and VL CDR3 of the 3G7HY antibody.
 28. The antibody ofclaim 1, wherein said isolated recombinant human antibody comprises VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the 3G7NYantibody.
 29. The antibody of claim 1, wherein said isolated recombinanthuman antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2,and VL CDR3 of the 3G7 antibody.
 30. The antibody of claim 1, whereinsaid isolated recombinant human antibody comprises VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2, and VL CDR3 of the SB2 antibody.
 31. Theantibody of claim 1, wherein said isolated recombinant human antibodycomprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 ofthe 2C8 antibody.
 32. The antibody of claim 1, wherein said isolatedrecombinant human antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1,VL CDR2, and VL CDR3 of the UA8kappa antibody.
 33. A pharmaceuticalformulation said formulation comprising: a pharmaceutically acceptableexcipient and an antibody according to claim
 1. 34. The formulationaccording to claim 33, wherein said formulation is formulated foradministration via a route selected from the group consisting of nasaladministration, rectal administration, intraperitoneal injection,intravascular injection, subcutaneous injection, transcutaneousadministration, and intramuscular injection.
 35. An immunoconjugatecomprising an antibody according to claim 1 attached to an effectorwherein said effector is selected from the group consisting of a secondantibody, a detectable label, a cytotoxin or cytostatic agent, aliposome containing a drug, a radionuclide, a drug, a prodrug, a viralparticle, a cytokine, and a chelate.
 36. The immunoconjugate of claim35, wherein said antibody is attached to a cytotoxic and/or cytostaticdrug.
 37. The immunoconjugate of claim 35, wherein said antibody isattached directly or through a linker to one or more of the following:said drug a lipid or liposome containing said drug; a polymeric drugcarrier comprising said drug; and a nanoparticle drug carrier comprisingsaid drug.
 38. The immunoconjugate of claim 37, wherein said drug is ananti-cancer drug.
 39. The immunoconjugate of claim 37, wherein said drugis selected from the group consisting of a microtubule inhibitor, aDNA-damaging agent, and a polymerase inhibitor.
 40. The immunoconjugateof claim 39, wherein the drug comprises a tubulin inhibitor.
 41. Theimmunoconjugate of claim 40, wherein the drug comprises: a drug selectedfrom the group consisting of an auristatin, Dolastatin-10, syntheticderivatives of the natural product Dolastatin-10, and maytansine or amaytansine derivative; a drug selected from the group consistingMonomethylauristatin F (MMAF), Auristatin E (AE), Monomethylauristatin E(MMAE), vcMMAE, and vcMMAF; or a maytansine selected from the groupconsisting of Mertansine (DM1), DM3, and DM4.
 42. The immunoconjugate ofclaim 39, wherein the drug comprises a drug selected from the groupconsisting of a calicheamicin, a calicheamicin analog, a duocarmycin, apyrrolobenzodiazepine, and a pyrrolobenzodiazepine dimer.
 43. Theimmunoconjugate of claim 42, wherein the drug comprises a duocarmycinselected from the group consisting of duocarmycin A, duocarmycin B1,duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D,duocarmycin SA, Cyclopropylbenzoindole duocarmycin, Centanamycin,Rachelmycin, Adozelesin, Bizelesin, and Carzelesin.
 44. Theimmunoconjugate of claim 42, wherein the drug comprise a drug selectedfrom the group consisting of Anthramycin, Mazethramycin, Tomaymycin,Prothracarcin, Chicamycin, Neothramycin A, Neothramycin B, DC-81,Sibiromycin, Porothramycin A, Porothramycin B, Sibanomycin, Abbeymycin,SG2000, and SG2285.
 45. The immunoconjugate of claim 37, wherein saiddrug is selected from the group consisting of auristatin, dolastatin,colchicine, combretastatin, and mTOR/PI3K inhibitors.
 46. Theimmunoconjugate of claim 37, wherein said drug is selected from thegroup consisting of fluorouracil, capecitabine,5-trifluoromethyl-2′-deoxyuridine, methotrexate sodium, raltitrexed,pemetrexed, cytosine Arabinoside, 6-mercaptopurine, azathioprine,6-thioguanine (6-TG), pentostatin, fludarabine phosphate, cladribine,floxuridine (5-fluoro-2), ribonucleotide reductase inhibitor (RNR),cyclophosphamide, neosar, ifosfamide, thiotepa,1,3-bis(2-chloroethyl)-1-nitosourea (BCNU),1,-(2-chloroethyl)-3-cyclohexyl-lnitrosourea, methyl (CCNU),hexamethylmelamine, busulfan, procarbazine HCL, dacarbazine (DTIC),chlorambucil, melphalan, cisplatin, carboplatin, oxaliplatin,bendamustine, carmustine, chloromethine, dacarbazine (DTIC),fotemustine, lomustine, mannosulfan, nedaplatin, nimustine,prednimustine, ranimustine, satraplatin, semustine, streptozocin,temozolomide, treosulfan, triaziquone, triethylene melamine, thioTEPA,triplatin tetranitrate, trofosfamide, uramustine, doxorubicin,daunorubicin citrate, mitoxantrone, actinomycin D, etoposide, topotecanHCL, teniposide (VM-26), camptothecin, belotecan, rubitecan,vincristine, vinorelbine tartrate, vindesine sulphate, paclitaxel,docetaxel, abraxane, ixabepilone, larotaxel, ortataxel, tesetaxel,vinflunine, retinoic acid, a retinoic acid derivative, vinblastine,interferon, tamoxifen, and taxol.
 47. A method of inhibiting the growthand/or proliferation of a cancer cell that expresses or overexpressesCD46, said method comprising: contacting said cancer cell with animmunoconjugate comprising an antibody according to claim 1 attached toan effector that has cytostatic and/or cytotoxic activity.
 48. Themethod of claim 47, wherein said cancer cell is selected from the groupconsisting of ovarian cancer, colorectal cancer, breast cancer, lungcancer, prostate cancer, kidney cancer, pancreatic cancer, mesothelioma,lymphoma, liver cancer, urothelial cancer, stomach cancer, multiplemyeloma, glioblastoma multiforme, glioma, neuroblastoma, and cervicalcancer.
 49. The method of claim 47, wherein said cancer cell is aprostate cancer cell.
 50. The method of claim 49, wherein said cancercell is a cell of a castration-resistant prostate cancer.
 51. The methodof claim 47, wherein said cell is a metastatic cell.
 52. The method ofclaim 51, wherein said metastatic cell is a bone metastasis, a livermetastasis, a bladder metastasis, and/or a lymph node metastasis. 53.The method of claim 47, wherein said cell is a solid tumor cell.
 54. Themethod of claim 47, wherein said effector comprises a radionuclideand/or a cytostatic drug.
 55. The method of claim 54, wherein saideffector comprises one or more of the following: a cytotoxic and/orcytostatic drug; a lipid or liposome containing a cytotoxic and/orcytostatic drug; a polymeric drug carrier comprising a cytotoxic and/orcytostatic drug; and a nanoparticle drug carrier comprising a cytotoxicand/or cytostatic drug.
 56. The method of claim 55, wherein said drug isan anti-cancer drug.
 57. The method of claim 56, wherein said drug isselected from the group consisting of auristatin, dolastatin,colchicine, combretastatin, and mTOR/PI3K inhibitors.
 58. The method ofclaim 56, wherein said drug is monomethyl auristatin F.
 59. The methodof claim 56, wherein said drug is selected from the group consisting offluorouracil, capecitabine, 5-trifluoromethyl-2′-deoxyuridine,methotrexate sodium, raltitrexed, pemetrexed, cytosine Arabinoside,6-mercaptopurine, azathioprine, 6-thioguanine (6-TG), pentostatin,fludarabine phosphate, cladribine, floxuridine (5-fluoro-2),ribonucleotide reductase inhibitor (RNR), cyclophosphamide, neosar,ifosfamide, thiotepa, 1,3-bis(2-chloroethyl)-1-nitosourea (BCNU),1,-(2-chloroethyl)-3-cyclohexyl-lnitrosourea, methyl (CCNU),hexamethylmelamine, busulfan, procarbazine HCL, dacarbazine (DTIC),chlorambucil, melphalan, cisplatin, carboplatin, oxaliplatin,bendamustine, carmustine, chloromethine, dacarbazine (DTIC),fotemustine, lomustine, mannosulfan, nedaplatin, nimustine,prednimustine, ranimustine, satraplatin, semustine, streptozocin,temozolomide, treosulfan, triaziquone, triethylene melamine, thioTEPA,triplatin tetranitrate, trofosfamide, uramustine, doxorubicin,daunorubicin citrate, mitoxantrone, actinomycin D, etoposide, topotecanHCL, teniposide (VM-26), camptothecin, belotecan, rubitecan,vincristine, vinorelbine tartrate, vindesine sulphate, paclitaxel,docetaxel, abraxane, ixabepilone, larotaxel, ortataxel, tesetaxel,vinflunine, retinoic acid, a retinoic acid derivative, vinblastine,interferon, tamoxifen, and taxol.
 60. The method of claim 47, whereinsaid administering comprises: administering parenterally; and/oradministering into a tumor or a surgical site.
 61. The method of claim47, wherein said antibody and/or immunoconjugate is administered as anadjunct therapy to surgery and/or radiotherapy.
 62. The method of claim47, wherein said antibody and/or immunoconjugate is administered inconjunction with another anti-cancer drug and/or a hormone.
 63. A methodof detecting a cancer cell of a cancer that expresses or overexpressesCD46, said method comprising: contacting said cancer cell with aimmunoconjugate comprising an antibody according to claim 1 attached toa detectable label; and detecting the presence and/or location of saiddetectable label where the presence and/or location is an indicator ofthe location and/or presence of a cancer cell.
 64. An isolatedrecombinant human antibody that specifically binds CD46 and isinternalized, via macropinocytosis, into a cell expressing oroverexpressing CD46, wherein said isolated recombinant human antibodycomprises: the variable light (VL) chain of the YS5 antibody and thevariable heavy (VH) chain of the YS5 antibody; or the variable light(VL) chain of the YS5F antibody and the variable heavy (VH) chain of theYS5F antibody; or the variable light (VL) chain of the YS5v1D antibodyand the variable heavy (VH) chain of the YS5v1D antibody; or thevariable light (VL) chain of the SB1HGNY antibody and the variable heavy(VH) chain of the SB1HGNY antibody; or the variable light (VL) chain ofthe YS12 antibody and the variable heavy (VH) chain of the YS12antibody; or the variable light (VL) chain of the 3G7RY antibody and thevariable heavy (VH) chain of the 3G7RY antibody; or the variable light(VL) chain of the YS6 antibody and the variable heavy (VH) chain of theYS6 antibody; or the variable light (VL) chain of the YS1 antibody andthe variable heavy (VH) chain of the YS1 antibody; or the variable light(VL) chain of the YS3 antibody and the variable heavy (VH) chain of theYS3 antibody; or the variable light (VL) chain of the YS4 antibody andthe variable heavy (VH) chain of the YS4 antibody; or the variable light(VL) chain of the YS8 antibody and the variable heavy (VH) chain of theYS8 antibody; or the variable light (VL) chain of the YS7 antibody andthe variable heavy (VH) chain of the YS7 antibody; or the variable light(VL) chain of the YS9 antibody and the variable heavy (VH) chain of theYS9 antibody; or the variable light (VL) chain of the YS10 antibody andthe variable heavy (VH) chain of the YS10 antibody; or the variablelight (VL) chain of the YS11 antibody and the variable heavy (VH) chainof the YS11 antibody; or the variable light (VL) chain of the 3G7HYantibody and the variable heavy (VH) chain of the 3G7HY antibody; or thevariable light (VL) chain of the 3G7NY antibody and the variable heavy(VH) chain of the 3G7NY antibody; or the variable light (VL) chain ofthe 3G7 antibody and the variable heavy (VH) chain of the 3G7 antibody;or the variable light (VL) chain of the SB2 antibody and the variableheavy (VH) chain of the SB2 antibody; or the variable light (VL) chainof the 2C8 antibody and the variable heavy (VH) chain of the 2C8antibody; or the variable light (VL) chain of the UA8kappa antibody andthe variable heavy (VH) chain of the UA8kappa antibody.